Use of inhibitors of indoleamine-2,3-dioxygenase in combination with other therapeutic modalities

ABSTRACT

The present invention provides improved treatment methods by the administration of both an inhibitor of indoleamine-2,3-dioxygenase in addition to the administration of an additional therapeutic agent.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/459,489, filed Apr. 1, 2003, and U.S. Provisional ApplicationSer. No. 60/538,647, filed Jan. 22, 2004. Both of these provisionalapplications are incorporated herein by reference in their entireties.

GOVERNMENT FUNDING

The present invention was made with government support under Grant Nos.K08 HL03395, 1R01 CA 103320, and 1R01 CA096651, awarded by the NationalInstitutes of Health. The Government may have certain rights in thisinvention.

BACKGROUND

The adaptive immune system must tailor the T cell repertoire so as notto respond to self-antigens. The classical model (reviewed by Nossal inCell 1994; 76:229-239) holds that autoreactive T cell clones are deletedin the thymus via a process of negative selection in which encounterwith antigen at the immature thymocyte stage triggers apoptosis,resulting in clonal deletion. Although the thymus undoubtedly provides amajor site of negative selection, there are difficulties with thismodel. First, it would seem unlikely that every developing T cell couldbe exposed to every self-antigen during its relatively brief transitthrough the thymus. Second, autoreactive T cells are empirically foundin the peripheral blood of normal, healthy hosts (Steinman, Cell 1995;80:7-10). This suggests the existence of additional means of tailoringthe T cell repertoire after the T cells have left the thymus, a processdesignated as peripheral tolerance.

The immune system of a tumor-bearing host often fails to respondprotectively against tumor antigens. Functionally, the host is toleranttoward the tumor (Smyth et al., Nat. Immunol., 2001; 2:293). This is notdue to a peculiarity of tumor antigens, because even highly immunogenicviral proteins become tolerizing when introduced on tumor cells(Staveley-O'Carroll et al., Proc. Natl. Acad. Sci. USA, 1998; 95:1178).Tumor-induced tolerance is actively created and is maintained in anongoing fashion (Sotomayor et al., Blood, 2001; 98:1070; and Cuenca etal., Cancer Res., 2003; 63:9007). Thus, tumors represent a striking andbiologically significant example of acquired peripheral tolerance(Pardoll, Ann. Rev. Immunol., 2003; 21:807). The molecular mechanisms bywhich this tolerance arises are currently unclear. This tolerance allowstumors to escape the host's normal immune surveillance and imposes afundamental barrier to successful clinical immunotherapy.

SUMMARY OF THE INVENTION

The present invention includes a method of treating a subject with acancer or an infection, the method including administering to thesubject an inhibitor of indoleamine-2,3-dioxygenase in an amounteffective to reverse indoleamine-2,3-dioxygenase-mediatedimmunosuppression, and administering at least one additional therapeuticagent wherein the administration of the inhibitor ofindoleamine-2,3-dioxygenase and the at least one additional therapeuticagent demonstrate therapeutic synergy. In some embodiments of themethod, the indoleamine-2,3-dioxygenase-mediated immunosuppression ismeditated by an antigen-presenting cell (APC).

In some embodiments of the method of the present invention, at least oneadditional therapeutic agent is an antineoplastic chemotherapy agent,including, for example, cyclophosphamide, methotrexate, fluorouracil,doxorubicin, vincristine, ifosfamide, cisplatin, gemcytabine, busulfan,ara-C, or combinations thereof.

In some embodiments of the method of the present invention, theadditional therapeutic agent is radiation therapy, including, forexample, localized radiation therapy delivered to the tumor and totalbody irradiation.

In some embodiments of the method of the present invention, theinhibitor of indoleamine-2,3-dioxygenase may be 1-methyl-tryptophan,β-(3-benzofuranyl)-alanine, β-(3-benzo(b)thienyl)-alanine, or6-nitro-D-tryptophan. In some embodiments, the inhibitor ofindoleamine-2,3-dioxygenase is a D isomer of an inhibitor ofindoleamine-2,3-dioxygenase, including, for example, the D isomer of1-methyl-tryptophan, the D isomer of β-(3-benzofuranyl)-alanine, the Disomer of β-(3-benzo(b)thienyl)-alanine, or the D isomer of6-nitro-D-tryptophan.

In some embodiments of the method of the present invention, the canceris melanoma, colon cancer, pancreatic cancer, breast cancer, prostatecancer, lung cancer, leukemia, brain tumors, lymphoma, sarcoma, ovariancancer, or Kaposi's sarcoma.

In some embodiments of the method of the present invention, the methodfurther includes bone marrow transplantation or peripheral blood stemcell transplantation.

In some embodiments of the method of the present invention, theinfection may be a viral infection, infection with an intracellularparasite, or an infection with an intracellular bacteria. In someembodiments, the viral infection is human immunodeficiency virus orcytomegalovirus. In some embodiments, the intracellular parasite may beLeishmania donovani, Leishmania tropica, Leishmania major, Leishmaniaaethiopica, Leishmania mexicana, Plasmodium falciparum, Plasmodiumvivax, Plasmodium ovale, or Plasmodium malariae. In some embodiments,the intracellular bacteria may be Mycobacterium leprae, Mycobacteriumtuberculosis, Listeria monocytogenes, or Toxplasma gondii.

In some embodiments of the method of the present invention, theadditional therapeutic agent is a vaccine. In some embodiments thevaccine may be an anti-viral vaccine, including, for example, a vaccineis against HIV. In some embodiments the vaccine is against tuberculosisor malaria. In some embodiments the vaccine is a tumor vaccine,including, for example, a melanoma vaccine. In some embodiments thetumor vaccine includes genetically modified tumor cells or a geneticallymodified cell line, including genetically modified tumor cells orgenetically modified cell line that have been transfected to expressgranulocyte-macrophage stimulating factor (GM-CSF). In some embodimentsthe vaccine includes one or more immunogenic peptides. In someembodiments the vaccine includes dendritic cells.

In some embodiments of the method of the present invention, theadditional therapeutic agent is a cytokine, including, for examplegranulocyte-macrophage colony stimulating factor (GM-CSF) orflt3-ligand.

The present invention also includes a method of augmenting the rejectionof tumor cells in a subject, the method including administering aninhibitor of indoleamine-2,3-dioxygenase and administering at least oneantineoplastic chemotherapeutic agent, wherein the rejection of tumorcells obtained by administering both the inhibitor ofindoleamine-2,3-dioxygenase and the antineoplastic chemotherapeuticagent is greater than that obtained by administering either theinhibitor of indoleamine-2,3-dioxygenase or the antineoplasticchemotherapeutic agent alone.

The present invention also includes a method of treating cancer, themethod including administering an inhibitor ofindoleamine-2,3-dioxygenase and administering at least oneantineoplastic chemotherapeutic agent, wherein cancer survival rateobserved by administering both the inhibitor ofindoleamine-2,3-dioxygenase and the antineoplastic chemotherapeuticagent is greater than the cancer survival rate observed by administeringeither the inhibitor of indoleamine-2,3-dioxygenase or theantineoplastic chemotherapeutic agent alone.

The present invention also includes a method of reducing tumor size orslowing tumor growth, the method including administering an inhibitor ofindoleamine-2,3-dioxygenase and administering at least oneantineoplastic chemotherapeutic agent, wherein the tumor size or tumorgrowth observed with the administration of both the inhibitor ofindoleamine-2,3-dioxygenase and the antineoplastic chemotherapeuticagent is less than the tumor size or tumor growth observed with theadministration of either the inhibitor of indoleamine-2,3-dioxygenase orthe antineoplastic chemotherapeutic agent alone.

The present invention also includes a method of augmenting rejection oftumor cells in a subject, the method including administering aninhibitor of indoleamine-2,3-dioxygenase and administering radiationtherapy, wherein the rejection of tumor cells wherein the rejection oftumor cells obtained by administering both the inhibitor ofindoleamine-2,3-dioxygenase and the radiation therapy is greater thanthat obtained by administering either the inhibitor ofindoleamine-2,3-dioxygenase or the radiation therapy alone.

The present invention also includes a method of treating cancer, themethod including administering an inhibitor ofindoleamine-2,3-dioxygenase and administering radiation therapy, whereinthe cancer survival rate observed by administering both the inhibitor ofindoleamine-2,3-dioxygenase and radiation therapy is greater than thecancer survival rate observed by administering either the inhibitor ofindoleamine-2,3-dioxygenase or radiation therapy alone.

The present invention also includes a method of reducing tumor size ortumor growth, the method including administering an inhibitor ofindoleamine-2,3-dioxygenase and administering radiation therapy, whereinthe tumor size or tumor growth observed with the administration of boththe inhibitor of indoleamine-2,3-dioxygenase and radiation therapy isless than the tumor size or tumor growth observed with theadministration of either the inhibitor of indoleamine-23-dioxygenase orradiation therapy alone.

The present invention also includes a method of treating an infection,the method including administering an inhibitor ofindoleamine-2,3-dioxygenase and administering at least one additionaltherapeutic agent, wherein a symptom of infection observed afteradministering both the inhibitor of indoleamine-2,3-dioxygenase and theadditional therapeutic agent is improved over the same symptom ofinfection observed after administering either the inhibitor ofindoleamine-2,3-dioxygenase or the additional therapeutic agent alone.IN some embodiments, the additional therapeutic agent is an antiviralagent, an antibiotic, an antimicrobial agent, a cytokine, or a vaccine.In some embodiments, the a symptom of infection observed may bereduction in viral load, increase in CD4⁺ T cell count, decrease inopportunistic infections, increased survival time, eradication ofchronic infection, or a combination thereof.

The present invention also includes a method of treating a subjectreceiving a bone marrow transplant or peripheral blood stem celltransplant by administering an inhibitor of indoleamine-2,3-dioxygenase.In some embodiments of the method, the inhibitor ofindoleamine-2,3-dioxygenase is administered in an amount effective toincrease the delayed type hypersensitivity reaction to tumor antigen,delay the time to relapse of post-transplant malignancy, increaserelapse free survival time post-transplant, and/or increase long-termpost-transplant survival. In some embodiments of the method theinhibitor of indoleamine-2,3-dioxygenase is administered prior to fullhematopoetic reconstitution.

DEFINITIONS

As used herein, the term “subject” represents an organism, including,for example, an animal. An animal includes, but is not limited to, ahuman, a non-human primate, a horse, a pig, a goat, a cow, a rodent,such as, but not limited to, a rat or a mouse, or a domestic pet, suchas, but not limited to, a dog or a cat.

As used herein “in vitro” is in cell culture, “ex vivo” is a cell thathas been removed from the body of a subject, and “in vivo” is within thebody of a subject.

As used herein, “treatment” or “treating” include both therapeutic andprophylactic treatments.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D. Expression of indoleamine 2,3-dioxygenase (IDO) byantigen-presenting cells (APC). In FIG. 1A human monocytes were analyzedwithout culture (fresh, n=12); cultured for 7 days in MCSF with IFN-γadded for the final 18 hours (Mφ+IFNγ, n=8); or cultured ingranulocyte-macrophage CSF+IL-4 (DCs) in BCS medium (n=34) or SFM(n=24). Upper row represents IDO versus CD 123; lower row representsCCR6 versus CD 123 on the same triple-stained cells. Negative controlfor IDO staining was the primary antibody preadsorbed with theimmunizing peptide. FIG. 1B represents immunophenotype of nonadherent(IDO⁺) dendritic cells (DCs) (dark lines) versus adherent cells (lightlines) from SFM cultures, matured with tumor necrosisfactor-α/IL-1β/IL-6/prostaglandin E₂. FIG. 1C represents morphology ofadherent (left) and nonadherent (right) cells (cytocentrifugepreparations, Wright's stain; scale bar, 10 μm). FIG. 1D representsimmunophenotype of MCSF-derived Mφs, gated separately on the CD123 (darklines) and CD123NEG (light lines) populations.

FIGS. 2A-2I. Effect of DC maturation on IDO expression. In FIG. 2Amonocyte-derived Mφs were analyzed with (left) or without (right) IFN-γadded for the final 18 hours. FIG. 2B represents DCs (BCS system), withor without IFN-γ for 18 hours. FIG. 2C represents DCs matured withantibody to CD40 on days 5 to 7, with or without IFN-γ for 18 hours.FIG. 2D represents DCs matured with antibody to CD40 on days 5 to 7,with or without IFN-γ for 18 hours, and with Il-10 added during thematuration step. FIG. 2E presents functional enzymatic activity.Depletion of tryptophan from the culture medium (expressed as apercentage of the starting tryptophan concentration in fresh medium, 25μM) by DCs with or without IFN-γ for 18 hours. Immature DCs (iDC),CD40-matured DCs (mDC), and CD40-matured DCs in the presence of IL-10(mDC/IL10) were generated, with or without IFN-γ activation, as in FIGS.2B-2D. In FIG. 2F allogeneic MLRs using enriched IDO⁺ DCs (nonadherentcells, SFM system, without added IFN-γ). DCs were either immature ormatured with antibody to CD40. APC/T cell ratio was 1:20. White bars,without 1MT; black bars, with 1 MT. FIG. 2G presents MLR with atitration of enriched IDO⁺ DCs (nonadherent cells, SFM system, no IFN-γ)matured with cytokine-containing supernatant from activated monocytes.Similar results were observed when DCs were matured with the cytokineregimen used in FIG. 1B. Responder T cell number, 5×10⁵ (highest APC/Tcell ratio was 1:10) without 1MT (triangles), and with 1MT (squares).FIG. 2H represents immunomagnetic sorting of CD123⁺ DCs from a mixed DCpreparation (BCS system, tumor necrosis factor-α (TNF-α) matured, noIFN-γ). Unfractionated (pre-sort), sorted CD123⁺ cells (greater than 80%purity), and CD123-depleted cells. APC/T cell ratio was 1:10. Whitebars, with 1 MT (DL-racemic mixture); black bars, without 1 MT. FIG. 2Irepresents adherent cells (less than 10% IDO⁺) from SFM cultures,matured with TNFα/IL1β/IL6/prostaglandin E₂, used as stimulators inallogeneic MLRs. White squares are without 1MT, black squares are with 1MT. For comparison, nonadherent (IDO⁺) cells from the same culture areshown without 1MT (triangles). Representative of six experiments.

FIGS. 3A-3E. Validation of polyclonal anti-IDO antibody. FIG. 3Apresents THP1 cell lysates, analyzed by western blot for IDO (12%SDS-PAGE gel, reducing conditions, blotted to PVDF membrane, blockedwith 10% dry milk in 0.25% Tween-20 Tris-buffered saline). In lane 1,anti-IDO primary antibody (10 ng/ml) was detected with anti-rabbitperoxidase secondary antibody (1:2000, Santa Cruz Biotechnology) andvisualized by chemiluminescence (ECL, Amersham). Lane 2, as in lane 1,but with 1 ug/ml immunizing peptide added to the primary antibody beforeuse. FIG. 3B presents MCSF-derived Mφs±IFNγ×18 hours, analyzed bywestern blot as in FIG. 3A. FIG. 3C represents immunoprecipitation fromTHP1 cell lysates. Cells were lysed with buffer (150 mM NaCl, 0.5%NP-40, 1 mM dithiothreitol, 2 mM EDTA, 50 mM Tris pH 7.5, plusinhibitors of proteases and phosphatases) and lysates incubated withanti-IDO antibody (1 ug, lane 1), or antibody plus immunizing peptide(lane 2). Immune-complexes were precipitated with protein G-agarose(Life Technologies), resolved by SDS-PAGE under reducing conditions, andanalyzed by silver stain. A single specific 45 kD band (arrow) wasresolved (just below the heavy chain band of the immunoprecipitatingantibody, IgGH). FIG. 3D presents 2D-gel electrophoresis followed bywestern blotting with anti-IDO antibody revealed a single majorimmunoreactive species in resting Mφs (left panel). Following activationwith IFNγ for 18 hours (right panel) two additional species (arrows)were detected by the anti-IDO antibody. MCSF-derived Mφs were lysed with8M urea and 4% CHAPS in Tris buffer, and 50 ug of protein subjected tofirst-dimension isoelectric focusing (Protean IEF system, BioRad) usingpH 3-10 ampholyte strips. Second-dimension reducing SDS-PAGE wasperformed (Mini-Protean system, BioRad) and gels were transferred toPVDF membrane and analyzed by western blot using anti-IDO antibody, asin FIG. 3A. FIG. 3E presents flow-cytometric analysis of MCSF-derivedMφs activated with IFNγ for 18 hours. Cells stained with anti-IDOantibody are shown on the left, while the same cells stained withantibody pre-adsorbed with the immunizing peptide are shown on theright.

FIGS. 4A-4B. Simultaneous analysis of T cell proliferation by thymidineincorporation and tryptophan concentration in the medium from the sameallogeneic MLR culture. FIG. 4A presents an analysis of T cellproliferation by thymidine incorporation from the allogeneic MLR culture(day 3 of activation). FIG. 4B presents an analysis of tryptophanconcentration in the medium by HPLC from the allogeneic MLR culture (day3 of activation). Stimulators were IDO+(nonadherent) DCs from SFMcultures. The ratio of DCs to T cells was low (1:500) in order toapproximate the density of APCs expected in normal lymphoid tissue.Under these condition there is little depletion of tryptophan from themedium (starting tryptophan concentration in RPMI medium of 25 uM), andthe concentration of tryptophan remains well above the level required tosupport T cell proliferation, indicated by the arrow in FIG. 4B.Nevertheless, there was still a significant component of IDO-mediatedsuppression, shown by the 3-fold enhancement of proliferation seen when1MT was added (FIG. 4A).

FIGS. 5A-5D. Suppression of T cell responses by tumor-draining LN cells.FIG. 5A shows dominant suppressor cells in TDLNs. Cells fromtumor-draining LN (DLN) and contralateral LNs (CLN) were harvested frommice with B78H1.GMCSF tumors on day 14 and used as stimulators in MLRs(the number of stimulator cells is shown in parentheses). Respondercells (5×10⁴ BM3 T cells) were the same in all groups. The left panelshows the absence of response when DLN cells were used as stimulators.The right panel shows mixing experiments, revealing that the absence ofresponse to the DLN cells was due to a dominant suppressor activitypresent in these cells. In all experiments, response to CLN stimulatorswas quantitatively comparable to control stimulators (normal LN cellsfrom non-tumor-bearing mice). FIG. 5B shows suppressive pDCs and Tregsin tumor-draining LNs. TDLN cells were sorted by 4-color flow cytometryinto CD11c⁺B220⁺ pDCs (1-2%) and CD4⁺CD25⁺ Tregs (2-3%), plus a thirdpopulation of all other cells (95-97%). Each fraction was used asstimulator cells in parallel MLRs, using the number of cells that wouldhave been present in 5×10⁴ of the original TDLN population. All MLRsreceived 5×10⁴ BM3 responders. FIG. 5C shows suppression by pDCs ismediated by IDO. TDLN cells were sorted into pDCs, Tregs, and thenon-suppressive “all other” cells, as in the previous panel (Tregs werediscarded). Each population was used as stimulators for BM3 responders,in duplicate MLRs with and without the IDO inhibitor 1MT. The left panelshows TDLN cells from a normal (IDO-sufficient) host show dominantsuppression by pDCs that is reversed by 1 MT (arrows). The right panelshows tumor-draining LN cells from IDO-knockout host, showing nosuppression, and no effect of 1 MT. FIG. 5D shows excess L-tryptophan(250 uM, 10×) abrogates suppression by pDCs in a fashion comparable to 1MT. Experimental design, as in previous panel. There was no effect of10× L-tryptophan when TDLN cells were derived from IDO-knockout mice.

FIGS. 6A-6C. Adoptive transfer of DCs from TDLNs creates profound localimmunosuppression in naive hosts. FIG. 6A shows antigen-drivenrecruitment of BM3 T cells into the LNs draining the site of adoptivetransfer. CD11c⁺DCs were purified from TDLNs (C57BL/6 hosts) andinjected subcutaneously into naive CBA mice. Recipient mice hadpreviously received 4×10⁷ naive BM3 splenocytes injected intravenously(“CBA+BM3” mice). Control CBA+BM3 recipients received normal CD11c⁺ DCsprepared from LNs of C57BL/6 mice without tumors (“Normal antigen(+)”),or from LNs of syngeneic CBA mice (“Antigen(−)”). After 10 days, the LNsdraining the site of the DC injection were harvested and stained forclonotype-specific BM3 TCR expression (left panel). Spleens from eachanimal were similarly stained (right panel). The number ofclonotype-positive CD8+ cells, expressed as a percentage of total CD8⁺ Tcells, are shown for each of the 3 different DC priming groups. Each barrepresents 4 pooled nodes. The absolute number of LN and spleen cellswas not significantly different between the 3 groups. FIG. 6B showsunresponsiveness of T cells primed with DCs from TDLNs. RecipientCBA+BM3 mice were primed with the 3 different types of DCs described inFIG. 6A. After 10 days, LN cells draining the site of DC injection wereharvested and used as responder cells in recall MLRs. A fixed number ofthe primed responder cells from each group (1×10⁵) were tested against atitration of stimulator cells (irradiated normal C57BL/6 splenocytes),as shown. In FIG. 6C recipient CBA+BM3 mice were primed with DCs fromTDLNs, or with DCs from normal C57BL/6 LNs. During the 10 day primingexposure, half of the recipient mice received 1-methyl-D-tryptophan (5mg/day) via implantable pellets; the group not labeled as receiving 1 MTreceived vehicle pellets alone. After 10 days, responder T cells wereharvested from LNs draining the site of each DC injection, and testedfor responsiveness in recall MLRs against a titration of irradiatedC57BL/6 splenocytes. All recipients were pre-loaded with identicalaliquots of BM3, and all recall MLRs were performed in parallel withidentical stimulators, so that comparison would be meaningful betweengroups.

FIGS. 7A-7C. Response to antigen introduced on IDO+DCs. FIG. 7A showssystemic awareness of antigen introduced on TDLN DCs is revealed by TCRdown regulation in spleen. The upper panel represents in vitrostimulation of BM3 T cells by cells from TDLN caused down-regulation ofTCR on BM3 T cells (detected by clonotype-specific antibody, gated onCD8+ cells). TCR down regulation was not induced by normal C57BL/6 LNstimulators. The lower panels represent adoptive transfer of CD11c⁺ DCswas performed as in FIG. 6A. After 10 days, LNs and spleen wereharvested from recipient CBA+BM3 mice stained for BM3 clonotype-specificTCR expression. The level of TCR expression is shown, gated on theclonotype-positive CD8⁺ BM3 cells in LN and spleen. Each histogramrepresents 4 pooled samples. FIG. 7B demonstrates TCR down regulation isdependent on functional IDO in the transferred DCs. B78H1×GM-CSF tumorswere grown in IDO-knockout mice (C57BL/6 background). DCs were sortedfrom TDLNs and used to prime recipient CBA+BM3 mice; control recipientsreceived TDLN DCs from wild-type C57BL/6 hosts, or normal C57BL/6 DCs.In the left panel, TCR expression on CD8+ BM3 T cells (measured as inFIG. 7A) showed little down regulation in the absence of IDO.Representative data from recipient LNs are shown; comparable resultswere obtained with T cells from spleen. In other experiments,administration of 1MT at the time of adoptive transfer alsosignificantly reduced TCR down regulation by TDLN DCs. The right panelconfirms that IDO-deficient TDLN DCs also did not suppress BM3 responsesin recall MLRs. FIG. 7C represents creation of secondary,IDO-independent immunosuppression following adoptive transfer. CBA+BM3recipients were primed with DCs from normal LNs (group #1), or with DCsfrom TDLNs (group #2), both from C57BL/6 mice. After 10 days, therecipient LNs draining the site of adoptive transfer were tested inrecall MLRs. In the left panel, mixing experiments showed that the lackof reactivity in T cells from mice receiving TDLN DCs was dominant,indicating active suppression. In the right panel, T cell proliferationwas not restored by 1 MT added to the MLR assays, suggesting thatrecipients had developed secondary, IDO-independent mechanisms ofimmunosuppression.

FIG. 8. IDO-mediated suppressor activity segregates with the CD19⁺subset of pDCs. Cells from TDLNs were sorted into five populations basedon expression of CD11c, B220, and CD19, as shown in the schematic attop. The three CD11c⁺DC fractions (labeled “A,” “13,” and “C”) were usedas stimulators in MLRs. Control MLRs were stimulated by B cells from thesame LN. Each MLR contained 2×10⁵ BM3 responder cells, plus a titrationof each type of stimulator cell (responders were in excess, stimulatorswere limiting). Parallel titrations were performed with and without 1MT,to demonstrate the IDO-mediated component of suppression. Only the CD19⁺pDCs (fraction A) mediated significant suppression.

FIGS. 9A-9D. The CD19⁺ DC subset displayed a phenotype consistent withmature plasmacytoid DCs. In FIG. 9A, TDLNs were stained by 4-color flowcytometry for CD11c versus CD19 versus the various markers shown. Thetotal CD11c⁺ DC population was gated into CD19⁺ and CD19^(NEG) subsets,and the expression of each marker shown for the two populations. Theisotype-matched negative control for each marker (gated on CD11c⁺ cells)is shown as the horizontal bar. Each histogram is representative of 4-12experiments with each marker. In FIG. 9B, cells from TDLNs were sortedinto 5 fractions, as shown in FIG. 8. RNA from each fraction wasanalyzed by real-time quantitative RT-PCR. RNA was added based onpre-determined equivalent amounts of gamma-actin message. Gels show theexpected molecular weight band for CDI9, pax5, and γ-actin after 27cycles of amplification. The numbers below each band give the relativeamount of message, quantitated against a standard curve of spleen RNA,normalized to the sorted B cell fraction (arbitrarily set equal to100%). FIG. 6C represents expression of maturation markers on CD19⁺versus CD19^(NEG) subsets of DCs from TDLNs, gated as in FIG. 9A.Maturation markers in the CD19^(NEG) population varied from experimentto experiment (an immature example is shown for comparison), but theCD19⁺ population was always mature. FIG. 9D represents expression ofcell-surface markers CD123 (IL3R-a) and CCR6 on the CD19⁺ and CDI9^(NEG)DC subsets. Cells were analyzed from TDLNs and from contralateral LNs ofthe same animals, as shown.

FIG. 10. Superior inhibitory activity of the D isomer of 1MT in an invitro bioassay. The ability of D- and L-1 MT to inhibit IDO-mediatedsuppression of human T cell proliferation was measured in allogeneicMLRs, using enriched IDO-expressing human DCs. Replicate MLRs receivedeach compound at the concentrations shown. Bars represent T cellproliferation at the end of the 5-day activation period.

FIGS. 11A-11E. Administration of 1-MT enhances immune-mediated hostanti-tumor effect when administered with radiation or cyclophosphamide.Mice were injected subcutaneously (SQ) with 4×10⁴ B16F10 cells. In FIG.11A 1MT (20 mg/day of a DL racemic mixture) was administered SQ bycontinuous-release copolymer pellets, with or without 500 cGy oftotal-body γ-irradiation. Control mice received vehicle pellets withoutdrug. In FIG. 11B 1MT was administered with cyclophosphamide (CPM) (150mg/kg, one dose). FIG. 11C represents 1MT and cyclophosphamide inrag1-knockout hosts. FIG. 11D represents the more potent pure D-isomerof 1 MT, given 5 mg/day, with cyclophosphamide.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

A newly recognized molecular mechanism contributing to peripheral immunetolerance is the immunoregulatory enzyme indoleamine 2,3-dioxygenase(IDO). Cells expressing the tryptophan-catabolizing enzyme IDO arecapable of inhibiting T cell proliferation in vitro and reducing T cellimmune responses in vivo (U.S. Pat. Nos. 6,451,840 and 6,482,416; Munnet al., Science 1998; 281:1191; Munn et al., J. Exp. Med. 1999;189:1363; Hwu et al., J. Immunol. 2000; 164:3596; Mellor et al., J.Immunol. 2002; 168:3771; Grohmann et al., J. Immunol. 2001; 167:708;Grohmann et al., J. Immunol. 2001; 166:277; and Alexander et al.,Diabetes 2002; 51:356).

IDO degrades the essential amino acid tryptophan (for reviews see Tayloret al., FASEB Journal 1991; 5:2516-2522; Lee et al., LaboratoryInvestigation, 2003; 83:1457-1466; and Grohmann et al., Trends inImmunology 2003; 24:242-248). Expression of IDO by humanmonocyte-derived macrophages (Munn et al., J. Exp. Med. 1999;189:1363-1372), human dendritic cells (Munn et al., Science 2002;297:1867-1870 and Hwu et al., J. Immunol. 2000; 164:3596-3599), andmouse dendritic cells (Mellor et al., J. Immunol. 2003; 171:1652-1655)allows these different antigen-presenting cells (APCs) to inhibit T cellproliferation in vitro. In vivo, IDO participates in maintainingmaternal tolerance toward the antigenically foreign fetus duringpregnancy (Munn et al., Science 1998; 281:1191-1193).

IDO has also been implicated in maintaining tolerance to self antigens(Grohmann et al., J. Exp. Med. 2003; 198:153-160), in suppressing T cellresponses to MHC-mismatched organ transplants (Miki et al.,Transplantation Proceedings 2001; 33:129-130), and in thetolerance-inducing activity of recombinant CTLA4-Ig (Grohmann et al.,Nature Immunology 2002; 3:985-1109). In these three systems, theimmunosuppressive effect of IDO can be blocked by the in vivoadministration of an IDO inhibitor, such as 1-methyl-tryptophan (alsoreferred to herein as 1-MT or 1MT).

The transfection of IDO into mouse tumor cell lines confers the abilityto suppress T cell responses both in vitro and in vivo (Mellor et al.,J. Immunol. 2002; 168:3771-3776). In a Lewis Lung carcinoma (LLC) model,administration of 1-MT significantly delayed tumor outgrowth (Friberg etal., International Journal of Cancer 2002; 101:151-155). The mousemastocytoma tumor cell line forms lethal tumors in naive hosts, but isnormally rejected by pre-immunized hosts. However, transfection of P815with IDO prevents its rejection by pre-immunized hosts (Uyttenhove etal., Nature Medicine 2003; 9:1269-1274). This effect was entirelydependent on the presence of an intact immune system and wassubstantially reversed, that is, tumor growth inhibited, by theconcomitant administration of 1-MT.

The selective recruitment of IDO⁺ APCs in the tumor-draining (sentinel)lymph nodes of patients with melanoma (Munn et al., Science 2002;297:1867-1870 and Lee et al., Laboratory Investigation 2003;83:1457-1466) indicates that tumors take advantage of theimmunosuppressive effect of IDO by recruiting a population ofIDO-expressing host APCs to present tumor antigens. Similar changes havebeen seen in breast carcinoma and other tumor-associated lymph nodes. Inmouse tumor models the IDO-expressing APCs in tumor-draining lymph nodesare phenotypically similar to a subset of dendritic cells recently shownto mediate profound IDO-dependent immunosuppressive in vivo (Mellor etal., Journal of Immunology 2003; 171:1652-1655). IDO-expressing APCs intumor-draining lymph nodes thus constitute a potent tolerogenicmechanism.

The present invention is based on the observation that theadministration of an inhibitor of indoleamine-2,3-dioxygenase to asubject suffering from a tumor or infection in combination withadministration of an additional therapeutic agent, results in animproved efficacy of therapeutic outcome when compared to thetherapeutic outcome observed with the administration of the inhibitor ofindoleamine-2,3-dioxygenase alone or the administration of theadditional therapeutic agent alone. While not intending to be limited toany single mechanism, the improved efficacy of therapeutic outcomeobserved may be due to a removal, reversal, or reduction of theimmunosuppressive effect of indoleamine-2,3-dioxygenase by theadministration an inhibitor of indoleamine-2,3-dioxygenase.

In some embodiments of the present invention, the improved efficacy oftherapeutic outcome observed with the administration of an inhibitor ofindoleamine-2,3-dioxygenase in combination with an additionaltherapeutic agent may be demonstrated by determination of thetherapeutic synergy. As used herein, a combination manifests“therapeutic synergy” if it is therapeutically superior to one or otherof the constituents used at its optimum dose (Corbett et al., CancerTreatment Reports, 66, 1187 (1982). In some embodiments of the presentinvention, the efficacy of a combination may be characterized by addingthe actions of each constituent.

The administration of both an inhibitor of indoleamine-2,3-dioxygenaseand at least one additional therapeutic agent can result in anaugmentation of the rejection of cells in a subject, wherein therejection of cells obtained by administering both the inhibitor ofindoleamine-2,3-dioxygenase and the additional therapeutic agent isgreater than that obtained by administering either the inhibitor ofindoleamine-2,3-dioxygenase or the additional therapeutic agent alone.As used herein the augmented rejection of cells includes an increasedlevel of immune system mediated rejection of the cells. As used herein,“cell” can include tumor cells and cells infected with an intracellularpathogen.

Likewise, the administration of both an inhibitor ofindoleamine-2,3-dioxygenase and at least one additional therapeuticagent can result in an increased cancer survival rate, a reduced orslowed tumor growth, the reduction in relapse to neoplasm, as inleukemia, a reduction in tumor progression, or a reduction in tumormetastasis, in comparison to that observed with the administration ofeither the inhibitor of indoleamine-2,3-dioxygenase or the additionaltherapeutic agent alone. As used herein, “increased cancer survivalrate,” “reduced tumor growth,” “slowed tumor growth,” “reduced relapseto neoplasm,” “reduced tumor progression,” or “reduced tumor metastasis”are as determined by established clinical standards.

The determination of immunosuppression mediated by an antigen presentingcell expressing indoleamine-2,3-dioxygenase (IDO) includes the variousmethods as described in the examples herein. T cell activation by anantigen-presenting cell and the stimulation of an immune response are asmeasured by standard methods well known in the immunological arts.

The enzyme indoleamine-2,3-dioxygenase (IDO) is well characterized (see,for example, Taylor et al., FASEB Journal 1991; 5:2516-2522; Lee et al.,Laboratory Investigation, 2003; 83:1457-1466; and Grohmann et al.,Trends in Immunology 2003; 24:242-248). Compounds that serve assubstrates or inhibitors of the IDO enzyme are also well known. Forexample, Southan et al., (Med. Chem Res., 1996; 343-352) utilized an invitro assay system to identify tryptophan analogues that serve as eithersubstrates or inhibitors of human IDO.

IDO inhibitors of the present invention include, but are not limited to,1-methyl-tryptophan, β-(3 benzofuranyl)-alanine,β-[3-benzo(b)thienyl]-alanine, 6-nitro-tryptophan, and derivativesthereof. Inhibitors of the IDO enzyme are readily commerciallyavailable, for example, from Sigma-Aldrich Chemicals, St. Louis, Mo. Aninhibitor of indoleamine-2,3-dioxygenase may be a L isomer of aninhibitor of indoleamine-2,3-dioxygenase, a D isomer of an inhibitor ofindoleamine-2,3-dioxygenase, or a racemic mixture of an inhibitor ofindoleamine-2,3-dioxygenase. In some embodiments, a preferred IDOinhibitor is 1-methyl-tryptophan, also referred to herein 1 MT.

In accordance with the present invention, an IDO inhibitor isadministered to a subject in combination with the administration of oneor more previously known treatment modalities. As used herein, the term“additional therapeutic agent” represents one or more agentsadministered in the previously known treatment modality. An additionaltherapeutic agent is not an inhibitor of IDO. For example, inhibitors ofIDO may be administered to a patient in combination with one or moreother modes of cancer treatment. Such additional therapeutic agentsinclude, but are not limited to, chemotherapy, radiation therapy,hormone therapy, surgical resection, treatment with an immunostimulatorycytokine, administration of an anti-tumor vaccine, antibody basedtherapies, whole body irradiation, bone marrow transplantation, andperipheral blood stem cell transplantation. The chemotherapeutic agentsused include, but are not limited to, cyclophosphamide, methotrexate,fluorouracil, doxorubicin, vincristine, ifosfamide, cisplatin,gemcytabine, busulfan (also known as 1,4-butanediol dimethanesulfonateor BU), ara-C (also known as 1-beta-D-arabinofuranosylcytosine orcytarabine), adriamycin, mitomycin, cytoxan, methotrexate, andcombinations thereof. The cytokines used include, but are not limitedto, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-8, IL-9, IL-10, IL-12,IL-18, IL-19, IL-20, IFN-α, IFN-β, IFN-γ, tumor necrosis factor (TNF),transforming growth factor-β (TGF-β), granulocyte colony stimulatingfactor (G-CSF), macrophage colony stimulating factor (M-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF)) (U.S. Pat.Nos. 5,478,556, 5,837,231, and 5,861,159), or Flt-3 ligand (Shurin etal., Cell Immunol. 1997; 179:174-184).

An IDO inhibitor may also be administered to a patient in combinationwith other modes of treatment for an infection. Such additionaltherapeutic agents may include, but are not limited to antiviral agents,antibiotics, antimicrobial agents, cytokines, and vaccines. Thecytokines used include, but are not limited to, IL-1α, IL-1β, IL-2,IL-3, IL-4, IL-6, IL-8, IL-9, IL-10, IL-12, IL-18, IL-19, IL-20, IFN-α,IFN-β, IFN-γ, tumor necrosis factor (TNF), transforming growth factor-β(TGF-β), granulocyte colony stimulating factor (G-CSF), macrophagecolony stimulating factor (M-CSF), granulocyte-macrophage colonystimulating factor (GM-CSF) (U.S. Pat. Nos. 5,478,556, 5,837,231, and5,861,159), or Flt-3 ligand (Shurin et al., Cell Immunol. 1997;179:174-184).

An IDO inhibitor may be administered to a patient receiving a vaccine.Such a vaccine may be an anti-viral vaccine, such as, for example, avaccine against HIV, or a vaccine against tuberculosis or malaria. Thevaccine may be a tumor vaccine, including, for example, a melanoma,prostate cancer, colorectal carcinoma, or multiple myeloma vaccine.Dendritic cells (DC) have the ability to stimulate primary T cellantitumor immune responses. Thus, a tumor vaccine may include dendriticcells. Dendritic cell vaccines may be prepared, for example, by pulsingautologous DCs derived from the subject with synthetic antigens, tumorlysates or tumor RNA, or idiotype antibodies, or transfection of DCswith tumor DNA, or by creating tumor cell/DC fusions (Ridgway, CancerInvest. 2003; 21(6):873-86). The vaccine may include one or moreimmunogenic peptides, for example, immunogenic HIV peptides, immunogenictumor peptides, or immunogenic human cytomegalovirus peptides (such asthose described in U.S. Pat. No. 6,251,399). The vaccine may includegenetically modified tumor cells, including genetically modified tumorcells to express granulocyte-macrophage stimulating factor (GM-CSF)(Dranoff, Immunol Rev. 2002; 188:147-54).

The administration of the IDO inhibitor may take place before, during,or after the administration of the other mode of therapy.

Inhibitors of IDO may be formulated as a composition. The compositionsof the present invention may be formulated in a variety of forms adaptedto the chosen route of administration. The formulations may beconveniently presented in unit dosage form and may be prepared bymethods well known in the art of pharmacy. Formulations of the presentinvention include, for example, pharmaceutical compositions including anIDO inhibitor and a pharmaceutically acceptable carrier. The phrase“pharmaceutically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of suchcompositions is well understood in the art. The formulations of thisinvention may include one or more accessory ingredients includingdiluents, buffers, binders, disintegrants, surface active agents,thickeners, lubricants, preservatives (including antioxidants), and thelike.

Formulations of an IDO inhibitor may further include one or moreadditional therapeutic agents. An additional therapeutic agent may be anantineoplastic chemotherapy agent, including, but not limited to,cyclophosphamide, methotrexate, fluorouracil, doxorubicin, vincristine,ifosfamide, cisplatin, gemcytabine, busulfan (also known as1,4-butanediol dimethanesulfonate or BU), ara-C (also known as1-beta-D-arabinofuranosylcytosine or cytarabine), adriamycin, mitomycin,cytoxan, methotrexate, or a combination thereof. Additional therapeuticagents include cytokines, including, but not limited to, macrophagecolony stimulating factor, interferon gamma, granulocyte-macrophagestimulating factor (GM-CSF), flt-3, an antibiotic, antimicrobial agents,antiviral agents, including, but not limited to, AZT, ddI or ddC, andcombinations thereof.

The tumors to be treated by the present invention include, but are notlimited to, melanoma, colon cancer, pancreatic cancer, breast cancer,prostate cancer, lung cancer, leukemia, lymphoma, sarcoma, ovariancancer, Kaposi's sarcoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma,multiple myeloma, neuroblastoma, rhabdomyosarcoma, primarythrombocytosis, primary macroglobulinemia, small-cell lung tumors,primary brain tumors, stomach cancer, malignant pancreatic insulanoma,malignant carcinoid, urinary bladder cancer, premalignant skin lesions,testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophagealcancer, genitourinary tract cancer, malignant hypercalcemia, cervicalcancer, endometrial cancer, and adrenal cortical cancer. As used herein,“tumor” refers to all types of cancers, neoplasms, or malignant tumorsfound in mammals.

The efficacy of treatment of a tumor may be assessed by any of variousparameters well known in the art. This includes, but is not limited to,determinations of a reduction in tumor size, determinations of theinhibition of the growth, spread, invasiveness, vascularization,angiogenesis, and/or metastasis of a tumor, determinations of theinhibition of the growth, spread, invasiveness and/or vascularization ofany metastatic lesions, and/or determinations of an increased delayedtype hypersensitivity reaction to tumor antigen. The efficacy oftreatment may also be assessed by the determination of a delay inrelapse or a delay in tumor progression in the subject or by adetermination of survival rate of the subject, for example, an increasedsurvival rate at one or five years post treatment. As used herein, arelapse is the return of a tumor or neoplasm after its apparentcessation, for example, such as the return of a leukemia.

Certain pathological conditions, such as parasitic infections, AIDS(caused by the human immunodeficiency virus (HIV) and latentcytomegaloviral (CMV) infections, are extremely difficult to treat sincethe macrophages act as reservoirs for the infectious agent. Even thoughthe cells are infected with by a foreign pathogen, they are notrecognized as foreign. The methods of the present invention may be usedto treat such pathological conditions including, but not limited to,viral infections, infection with an intracellular parasite, andinfection with an intracellular bacteria. Viral infections treatedinclude, but are not limited to, infections with the humanimmunodeficiency virus (HIV) or cytomegalovirus (CMV). Intracellularbacterial infections treated include, but are not limited to infectionswith Mycobacterium leprae, Mycobacterium tuberculosis, Listeriamonocytogenes, and Toxplasma gondii. Intracellular parasitic infectionstreated include, but are not limited to, Leishmania donovani, Leishmaniatropica, Leishmania major, Leishmania aethiopica, Leishmania mexicana,Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, andPlasmodium malariae.

The efficacy of treatment of an infection may be assessed by any ofvarious parameters well known in the art. This includes, but is notlimited to, a decrease in viral load, an increase in CD4⁺ T cell count,a decrease in opportunistic infections, eradication of chronicinfection, and/or increased survival time.

An inhibitor of IDO may be administered to a subject following bonemarrow transplantation or a peripheral blood stem cell transplantation.The efficacy of such an administration may be assessed by any of avariety of parameters well known in the art. This includes, for example,determinations of an increase in the delayed type hypersensitivityreaction to tumor antigen, determinations of a delay in the time torelapse of the post-transplant malignancy, determinations of an increasein relapse free survival time, and/or determinations of an increase inpost-transplant survival. The IDO inhibitor may be administered to thesubject prior to full hematopoetic reconstitution or prior to recoveryfrom lymphopenia.

The inhibitors of the present invention can be administered by anysuitable means including, but not limited to, for example, oral, rectal,nasal, topical (including transdermal, aerosol, buccal and sublingual),vaginal, parenteral (including subcutaneous, intramuscular, intravenousand intradermal), intravesical, or injection into or around the tumor.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, intraperitoneal, and intratumoraladministration. In this connection, sterile aqueous media that can beemployed will be known to those of skill in the art in light of thepresent disclosure (see for example, “Remington's PharmaceuticalSciences” 15th Edition). Some variation in dosage will necessarily occurdepending on the condition of the subject being treated. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity, andgeneral safety and purity standards as required by the FDA.

For enteral administration, the inhibitor will typically be administeredin a tablet or capsule, which may be enteric coated, or in a formulationfor controlled or sustained release. Many suitable formulations areknown, including polymeric or protein microparticles encapsulating drugto be released, ointments, gels, or solutions which can be usedtopically or locally to administer drug, and even patches, which providecontrolled release over a prolonged period of time. These can also takethe form of implants. Such implant may be implanted within the tumor.

Therapeutically effective concentrations and amounts may be determinedfor each application herein empirically by testing the compounds inknown in vitro and in vivo systems, such as those described herein;dosages for humans or other animals may then be extrapolated therefrom.

An IDO inhibitor may be administered at once, or may be divided into anumber of smaller doses to be administered at intervals of time. It isunderstood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions andmethods.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Potential Regulatory Function of Human DendriticCells Expressing Indoleamine 2,3-Dioxygenase

This example describes a subset of human APCs that express indoleamine2,3-dioxygenase (IDO) and inhibit T cell proliferation in vitro.IDO-positive APCs constituted a discrete subset identified bycoexpression of the cell-surface markers CD123 and CCR6. In thedendritic cell (DC) lineage, IDO-mediated suppressor activity waspresent in fully mature as well as immature CD 123⁺ DCs. IDO⁺ DCs couldalso be readily detected in vivo, indicating that these cells representa regulatory subset of APCs in humans.

Using an IDO-specific antibody (FIG. 3), it was shown by flow cytometrythat fresh human monocytes expressed low to undetectable levels of theIDO protein (FIG. 1A). Monocyte-derived macrophages (Mφs) up-regulatedIDO upon activation with interferon-γ (IFN-γ) (Munn et al., J. Exp. Med,1999; 189:1363). Expression of IDO in these cells was confined to aparticular subset of cells coexpressing CD123 (the interleukin-3 (IL-3)receptor α chain] and the chemokine receptor CCR6 (FIG. 1A). Similarly,monocyte-derived DCs (Sallusto and Lanzavecchia, J. Exp. Med., 1994;179:1109) expressed IDO, which was also confined to a CD 123⁺, CCR6⁺subset.

Because serum factors are known to influence DC maturation (Romani etal., J. Immunol. Methods, 1996; 196:137), DCs were derived in bothbovine calf serum (BCS)-based medium and serum-free medium (SFM). Bothsystems yielded IDO⁺ DCs with the same phenotype, but greater than 90%of the IDO^(NEG) cells in SFM were tightly adherent. This allowed facileenrichment of the nonadherent IDO⁺ population to greater than 90%purity. The IDO⁺ cells expressed cell-surface markers CD14^(NEG), CD83⁺,CD80⁺, CD86^(HI), and HLA-DR^(HI) (FIG. 1B), morphology consistent withmature DCs (FIG. 1C). Adherent cells lacked CD83 and displayed residuallevels of CD14, consistent with an immature or transitional phenotype.Although IDO⁺ cells in DC cultures expressed DC-specific lineagemarkers, and the IDO⁺ Mφs expressed Mφ-lineage markers (FIG. 1D), inboth types of cells the IDO⁺ subset could be specifically identified byexpression of CD123 and CCR6.

As shown previously (Munn et al., J. Exp. Med., 1999; 189:1363), restingmacrophage colony-stimulating factor (MCSF)-derived Mφs did not expresshigh concentrations of IDO until they received a triggering signal suchas IFN-γ (FIG. 2A). In contrast, IDO could be detected constitutively inCD123⁺ DCs (FIG. 2B). However, activation with IFN-γ was still requiredfor expression of functional enzymatic activity FIG. 2E), which suggeststhat the IDO protein could exist in both enzymatically active andinactive states. Posttranslational regulation of enzymatic activity(constitutive expression of enzyme protein but with additional signalsbeing required for functional activity) is a feature of many regulatoryenzymes. Expression of IDO protein without enzymatic activity has beendescribed in murine DCs (Fallarino et al., Int. Immunol., 2002; 14:65).However, the mechanism by which IDO might exist in distinct functionalstates remains to be determined. Because the maturational status of DCsmay affect a number of functional attributes of these cells, it wasdetermined whether maturation affected IDO expression by CD123⁺ DCs.Although maturation itself had no effect on the constitutive (basal)expression of IDO protein, subsequent activation of mature DCs withIFN-γ resulted in complete down-regulation of IDO. This was a consistentobservation in 16 experiments with 10 different donors and was confirmedby flow cytometry (FIG. 2C), enzymatic activity (FIG. 2E), and mRNA.

Interleukin 10 (IL-10) is a regulatory cytokine that has been associatedwith the development of tolerogenic DCs (Steinbrink et al., J. Immunol.,1997; 159:4772). The presence of IL-10 during maturation preventedIFN-γ-induced down-regulation of IDO, resulting in sustained expressionof functional IDO even in mature, IFN-γ-activated DCs (FIGS. 2D and 2E).Similar results were observed when transforming growth factor-β waspresent during maturation. Taken together, these data raised thepossibility that expression of IDO by mature DCs might be determined bythe prevailing regulatory influences during maturation.

DCs were next tested for their ability to stimulate T cells inallogeneic mixed-leukocyte reactions (MLRs). In FIGS. 2B-2E, recombinantIFN-γ was added to simulate signals from activating T cells (Munn etal., J. Exp. Med., 1999 189:1363), but for MLRs the DCs received noexogenous IFN-γ. Immature DCs, selected and enriched to >90% purity forIDO expression (FIG. 1B), stimulated very little T cell proliferation(FIG. 2F). However, in most donors proliferation could be significantlyenhanced by addition of 1-methyl-D-tryptophan (1MT), a competitiveinhibitor of IDO. After maturation, enriched IDO⁺ DCs displayed one oftwo patterns: in 4 of 45 experiments the mature DCs lost theirIDO-mediated inhibitory activity (FIG. 2F), whereas in 41 of 45experiments they maintained potent inhibitory activity despitematuration, which was reversed by the addition of 1 MT (FIG. 2G).Continued expression of IDO by mature DCs in the latter experiments wasconfirmed by flow cytometry on MLR cultures and by measurement oftryptophan and kynurenine in supernatants. The two different patternsobserved in MLR were suggestive of the different patterns observed whenmature DCs were tested in isolation (FIGS. 2C and 2D).

The experiments in FIG. 2G were performed with highly enriched IDO⁺ DCsfrom SFM cultures. In contrast, BCS medium yielded a mixture ofnonadherent IDO⁺ and IDO^(NEG) cells, with IDO^(NEG) cells typically inthe majority. Under these conditions, T cell activation predominated,and 1 MT had little detectable effect (FIG. 2H). However, when the IDO⁺DCs were enriched from such mixtures by sorting for CD123 expression,they displayed inhibitory activity comparable to the IDO⁺ DCs from SFM(FIG. 2H). To verify the specificity of 1 MT as an inhibitor of IDO, 1MT was added to MLRs containing APCs that did not express inhibitoryamounts of IDO (adherent cells from SFM cultures, less than 10% IDO).Under these conditions, T cell proliferation was not inhibited, and 1 MThad no effect on T cell proliferation (FIG. 2I).

The staining of tissue from fifteen non-inflamed tonsils removed inroutine tonsillectomy showed scattered IDO+ cells in germinal center andT cell regions, indicating that in vivo, few IDO⁺ cells are detected innormal lymphoid tissue. However, human tonsils displaying features ofchronic inflammation often possessed intense focal infiltrates of IDO⁺cells, which were morphologically distinct from Ham56⁺ macrophages(Orenstein and Wahl, Ultrastruct. Pathol., 099; 23:79) or S100⁺interdigitating DCs (Cochran et al., Mod. Pathol., 2001; 14:604). SomeIDO⁺ cells coexpressed CD83, a marker of mature DCs, and some expressedCD123 and CCR6 (FIG. 3B). However, not all CD83⁺ (or CD 123⁺ or CCR6⁺)cells expressed IDO, and no single marker identified all IDO⁺ cells,which suggests that IDO may be expressed by more than one population invivo. Analyses of regional and sentinel (first draining) lymph nodestaken from patients with malignant melanoma revealed that 25 of 66patients had one or more nodes with abnormal accumulation of IDO⁺ cells.

For these studies, lymph nodes were analyzed from 26 patients withmalignant melanoma at the Medical College of Georgia; 13 of 26 patientswere found to have at least one node (often more than one) with markedlyabnormal accumulation of IDO⁺ cells (3+ or 4+ on a 4+ scale,independently graded by 3 pathologists). The IDO⁺ cells weremorphologically monocytic or plasmacytoid in appearance, and were foundinfiltrating extensively throughout the lymphoid regions, typicallyconcentrating in the interfollicular and T cell zones. Accumulationswere observed around blood vessels, at the margins of medullary sinuses,and the interface between lymphoid tissue and tumor metastases.Recruitment of IDO⁺ dendritic cells to specific regional lymph nodes wasalso seen in smaller series of patients with carcinoma of the breast,lung, colon and pancreas (comprising all of the other histologiesexamined).

In a separate study, sentinel lymph nodes were obtained from patientswith malignant melanoma. Each node was documented to be the initialtumor-draining lymph node by in vivo lymphoscintigraphy, and all werenegative for metastatic tumor by conventional pathologic studies. Twelveof these 40 sentinel nodes were found to have abnormal accumulation ofIDO⁺ cells (grades 1+ to 4+). In many of these patients, accumulation ofIDO⁺ cells in the sentinel node occurred before overt metastasis.Similar accumulation of IDO⁺ cells was found in nodes from patients withbreast, colon, lung, and pancreatic cancers.

In this example, a subset of human monocyte-derived DCs that use IDO toinhibit T cell proliferation in vitro is described. In both DC and Mlineages, IDO⁺ cells could be characterized by coexpression of CD123 andCCR6 (despite the expression of otherwise distinct lineage-specificmarkers), which suggests that the IDO⁺ population may represent adiscrete subset of professional APCs. IDO⁺ DCs expressed majorhistocompatibility complex class II and costimulatory molecules and wereeffective stimulators of T cell proliferation when IDO was blocked by1MT, which suggests that these cells could act as competent APCs. Thismay reflect a regulatory subset of APCs specialized to causeantigen-specific depletion (Munn et al., J. Immunol 1996; 156:523) orotherwise negatively regulate the responding population of T cells. Inlight of the finding that large numbers of such cells are present in aproportion of tumor-draining lymph nodes, it appears that IDO⁺ APCs mayparticipate in the state of apparent immunologic unresponsivenessdisplayed by many cancer patients toward tumor-associated antigens.However, the extent to which IDO-expressing APCs might influenceimmunologic unresponsiveness in vivo remains to be determined.

Polyclonal Antibody Preparation and Validation

The peptide LIESGQLRERVEKLNMLC (SEQ ID NO:1) was prepared based on theGenBank sequence of human IDO (GenBank Accession Number M34455; Dai andGupta, Biochem. Biophys. Res. Commun., 1990; 168:1-8) and conjugated tokeyhole limpet cyanogen. Rabbits were immunized with conjugated peptidein complete Freund's adjuvant and boosted three times in incompleteFreund's adjuvant (all antibody preparation and affinity purificationsteps were performed by QCB/BioSource International). This peptide gavethe best results out of several sequences screened for their ability todetect IDO in formalin-fixed paraffin-embedded tissue and flowcytometry. Using THP1 monocytic leukemia cell lysates, the antibodydetected a single band of the expected 45 kD molecular mass (Dai andGupta, Biochem. Biophys. Res. Commun., 1990; 168:1-8) by western blot(FIG. 3A). Immunoreactivity in western blot was blocked bypre-adsorption of the antibody with an excess of the immunizing peptide(FIG. 3A). The 45 kD band was inducible by IFNγ in humanmonocyte-derived Mφs (FIG. 3B), consistent with previous western blotstudies by others (Thomas et al., J. Immunol., 2001; 166:6332; and Grantet al., J. Virol., 2000; 74:4110). Immunoprecipitation of THP1 celllysates yielded a single 45 kD protein band on silver stain, which wasnot precipitated in the presence of the immunizing peptide (FIG. 3C).The antibody detected an IFNγ-inducible antigen by flow cytometry inmonocyte-derived macrophages, and this signal was reduced by greaterthan 95% by pre-adsorption of the antibody with the immunizing peptide(FIG. 3D). By immunohistochemistry on formalin-fixed, paraffin-embeddedspecimens of human placenta, the antibody detected an antigenspecifically localized to syncytiotrophoblast (FIG. 3E).Syncytiotrophoblast was used for validation′because it is an unambiguouscell type that has been previously shown to express IDO byimmunohistochemistry (Kamimura et al., Acta. Med Okayama, 1991; 45:135),which has been independently confirmed using enzymatic-activity assayson highly purified placental cell fractions (Kudo and Boyd, Biochem.Biophys. Acta, 2000; 1500:119). Reactivity in immunohistochemistry wasfully blocked by pre-adsorption of the antibody with the immunizingpeptide (FIG. 3E).

Distinction of IDO⁺ Cells from CD123⁺ Plasmacytoid DCs

CD123 is also found on “plasmacytoid” DCs or pre-DC2 cells. However, theCD123 expression observed on the IDO⁺ subset of monocyte-derived DCs wasat the lower level described on myeloid DCs (Cella et al., Nat. Med.,1999; 5:919), not the 10- to 100-fold higher levels that we observed onplasmacytoid DCs. The IDO⁺ cells also expressed myeloid markers (CD11b,CD11c) and did not express the pre-DC2 marker BDCA2 (Dzionek et al., J.Immunol., 2000; 165:6037). Plasmacytoid dendritic cells (CD123⁺CD11c^(NEG) cells) showed no detectable expression of IDO (fresh orfollowing activation with IFNγ). The CD123⁺ IDO⁺ subset ofmonocyte-derived DCs and Mφs co-expressed CCR6. While CD123 wasexpressed constitutively, CCR6 was inducible, and was expressed underthe same conditions in which IDO protein was induced. Thus, inMCSF-derived Mφs CCR6 required induction by IFNγ, whereas in DCs CCR6was constitutively expressed on CD123⁺ DCs.

Monocyte Isolation and Culture

Human monocytes (typically greater than 95% purity) were isolated byleukocytapheresis and counterflow elutriation as described (Munn et al.,J. Exp. Med., 1999; 189:1363), then cryopreserved in replicate aliquots.Monocyte-derived Mφs were cultured in RPMI-1640 medium supplemented with10% bovine calf serum (Hyclone) (“BCS system”) and received MCSF (200units/ml, gift of Genetics Institute) on day 0 (Munn et al., J. Exp.Med., 1999; 189:1363). Monocyte-derived DCs were cultured in 100 mmpetri dishes in either BCS medium (Sallusto and Lanzavecchia, J. Exp.Med., 1994; 179:1109) or in serum-free medium (X-vivo 15, BioWhitaker,“SFM system”) (Chen et al., Blood, 1998; 91:4652). SFM yielded asomewhat higher proportion of IDO⁺ cells, but the phenotype and functionof the cells was identical. DCs received GMCSF (50 ng/ml, R&DSystems)+IL4 (50 ng/ml, R&D Systems) on days 0, 2 and 4. For experimentswhere CCR6 expression was of interest, cultures received a single doseof GMCSF+IL4 (100 ng/ml each) on day 0, which gave higher expression ofCCR6 (Yang et al., J. Immunol., 1999; 163:1737). Non-adherent DCs wereharvested by aspiration; adherent cells and MCSF-derived macrophageswere harvested with 5 mM EDTA. For maturation studies, cells weretreated for the final 48 hours with one of the following: 0.5 ug/mlactivating anti-CD40 antibody (Mabtech); a cytokine cocktail comprisingTNFα (1100 units/ml, Pharmingen), IL1β (1870 units/ml, Pharmingen), IL6(1000 units/ml, Pharmingen) and PGE2 (1 ug/ml, Sigma) (Jonuleit et al.,Eur. J. Immunol., 1997; 27:3135) (12); or 50% v/v conditioned medium(X-vivo 15) from activated monocytes (allowed to adhere overnight topetri dishes coated with immobilized IgG (Cappel ICN) (Reddy et al.Blood, 1997; 90:3640). In some experiments, DCs also received IL10 (R&DSystems, 100 ng/ml) with the maturation stimuli. For activation studies,cells received 100 units/ml IFNγ (Genentech) during the final 18 hoursof culture.

Flow Cytometry

All antibodies and reagents were from BD-Pharmingen unless specified.Fresh whole blood was stained using BD FACS Lysing Solution. Forcultured cells, nonadherent cells were pooled with adherent cells(harvested with 5 mM EDTA in PBS for 10 minutes) prior to analysis,unless otherwise specified. Cells were triple-stained withanti-CD123-biotin (clone 7G3) followed by streptavidin-perCP, plusanti-CD11c-allophycocyanin (clone S-HCL-3) or anti-CCR6-fluorescein(clone 53103.111, R&D systems). CCR6 results were confirmed using asecond antibody (11A9, BD-Pharmingen). After fixation andpermeablization (Cytofix/Cytoperm), cells were stained with rabbitanti-IDO antibody followed by PE-labeled anti-rabbit secondary antibody(Jackson Immunoreasearch, cross-adsorbed against mouse, human and bovineIgG). For all experiments, the negative control for IDO staining was theanti-IDO antibody pre-adsorbed with a 50-fold molar excess of theimmunizing peptide. Dendritic cells and Mφs were gated on forward andside scatter to exclude contaminating lymphocytes and debris. Forphenotyping experiments, cells were stained without permeablization,using a multicolor panel of CD123 versus various markers (all fromBD-Pharmingen).

Mixed-Leukocyte Reactions

Dendritic cells were mixed with 5×10⁵ allogeneic lymphocytes (80-85% Tcells, balance B cells and NK cells, less than 1% monocytes) in 250 ulof MLR culture medium (10% fetal calf serum in RPMI-1640). Replicatewells received 1-methyl-tryptophan (Aldrich) at a final concentration of200 uM at the time of T cell addition, or buffer control. Unlessotherwise specified, the D-isomer of 1MT was used. After five days,proliferation was measured by four hour thymidine incorporation assay.“V”-bottom culture wells (Nunc) were used in MLR assays to maximizecell-cell contact. It was found that this geometry gave significantlygreater sensitivity for measuring suppression, as compared toflat-bottom wells. However, V-bottom wells were not a prerequisite fordetecting suppression (e.g., all of the experiments in both Munn et al.,J. Exp. Med., 1999; 189:1363; and Mellor et al., J. Immunol., 2002;168:3771 used flat-bottom wells). The V-bottom geometry itself was notsuppressive, as shown by the brisk T cell proliferation observed whenIDO was inhibited by 1MT (FIG. 2G), or when IDO^(NEG) APCs were used asstimulators (FIG. 2I). Tryptophan depletion in these cultures is shownin FIG. 4. The data represented in FIG. 4 indicate that inhibition ofproliferation did not depend on artificially high concentrations of APCsor global depletion of tryptophan from the entire culture medium.

However, these experiments were still consistent with a localizeddepletion of tryptophan (i.e., occurring within the immediate zone ofcontact between DCs and T cells), since addition of supra-physiologiclevels of tryptophan to the culture medium (250 uM) completely reversedthe DC-mediated inhibition of T cell proliferation, as has beenpreviously described (Munn et al., J. Exp. Med., 1999; 189:1363).

Immunohistochemistry

For paraffin blocks, sections were deparaffinized, treated for 8 minuteswith proteinase K (Dako), and stained with rabbit anti-human IDOantibody (5 ug/ml in phosphate-buffered saline with 0.05% Tween-20 and10% goat serum). Detection was via secondary antibody conjugated toalkaline phosphatase (LSAB2-rabbit kit, Dako) with Fast Red ornitro-blue tetrazolium chromogen. In all experiments, negative controlsconsisted of the anti-IDO antibody neutralized with a molar excess ofthe immunizing peptide. For two-color immunofluorescent staining, frozensections of human tonsil were fixed for 10 minutes in 10% formalin, thenprimary antibodies were applied against IDO (0.05 ug/ml) and either CD83(Immunotech), CD123 (Santa Cruz) or CCR6 (R&D Systems). IDO was detectedwith rabbit-specific secondary antibody conjugated to Alexa-488(Molecular Probes); mouse antibodies were detected with species-specificsecondary antibody conjugated to Alexa-568 (all secondary antibodieswere cross-adsorbed for multiple labeling). Some CD4⁺ T cells showedlow-level staining for IDO, consistent with previous reports (Curreli etal., J. interferon Cytokine Res., 2001; 21:431). This T cell stainingwas minimal by the less-sensitive immunohistochemistry technique, andwith immunofluorescence could be unambiguously distinguished from thehigh-level IDO expression seen in APCs through the use of two-colorstaining.

Use of Recombinant IFNγ

When DCs or Mφs were tested in isolation (i.e., without T cells, as inFIGS. 2A-2E) recombinant human IFNγ was added during the final 18 hoursof culture), in order to simulate the activating signals that wouldnormally be delivered by activating T cells to APCs duringantigen-presentation (Munn et al., J. Exp. Med., 1999; 189:1363). Thisallowed the identification of 3 stages at which IDO expression could beregulated in DCs; differentiation, maturation, and the final activationstep (e.g., by IFNγ). Simulating this final step proved critical inelucidating the differential regulation of IDO in mature versus immatureDCs (see FIGS. 2B-2D). In this system, IFNγ was found to be capable ofboth up-regulating and down-regulating IDO, depending the maturationstatus of the DCs. This dual role for IFNγ is consistent with theliterature, which has shown roles for IFNγ both as a pro-inflammatorycytokine, and as a participant in tolerance and negative regulation(Konieczny et al., J. Immunol., 1998; 160:2059). When DCs were to beused in MLRs they were not exposed to IFNγ during culture, nor wasrecombinant IFNγ added to the MLRs. Thus, any activating signalsinfluencing expression of IDO in MLRs would be derived physiologically,from the T cells themselves.

Two Patterns of IDO Expression in Mature DCs

FIG. 2C and FIG. 2D tested mature DCs in isolation, using IFNγ tosimulate signals from T cells. While the cytokine milieu in actual MLRsis more complex than this simplified system, the pattern observed inFIG. 2F (loss of inhibitory activity for T cells when DCs were matured)was consistent with the pattern shown in FIG. 2C (down-regulation of IDOwhen mature DCs were activated with IFNγ). In contrast, the patternshown in FIG. 2G (maintenance of inhibitory activity in MLR despitematuration) was consistent with FIG. 2D (expression of functional IDO inmature DCs). It is currently unknown what factors in the MLRs favoredmaintenance of IDO-mediated suppression in the large majority ofexperiments. One potentially significant variable could be theproduction of regulatory cytokines such as IL10 by the activated Tcells. This issue will require further investigation to elucidate.However, the key observation arising from FIGS. 2D and 2G is that theCD123⁺ subset of DCs has the potential for IDO-meditated inhibition of Tcells even when mature.

Donor-to-Donor Variability

Inhibition of T cell proliferation by immature DCs was seen in 10/10experiments using seven different donors. In seven of these tenexperiments, T cell proliferation was significantly enhanced by 1MT.With mature DCs, inhibition of T cell proliferation was observed in12/13 experiments (six different donors) using sorted CD123⁺ DCs (BCSsystem) as shown in FIG. 2H. Also with mature DCs, inhibition wasobserved in 28/32 experiments (nine different donors) using non-adherentIDO-enriched cells (SFM system) as shown in FIG. 2G. In four experimentsusing mature DCs (four donors), the DCs showed loss of inhibition uponmaturation, the pattern shown in FIG. 2F. However, when two of thesefour donors were tested in multiple experiments it was found that byusing different conditions (different maturation regimen, differentresponder T cells), these donors could be induced to show inhibitionafter maturation. Thus, the majority of donors showed IDO-mediatedinhibition by mature DCs, and this was subject to influence by theconditions prevailing during maturation and MLR.

Use and Limitations of 1MT

In all experiments, the D-isomer of 1 MT was used unless otherwisespecified, because it gave better reversal of suppression with lesstoxicity than the DL-racemic mixture used previously (Munn et al.,Science, 1998; 281:1191). As previously published (Munn et al., J. Exp.Med., 1999; 189:1363), 1 MT is not an efficient inhibitor of IDO (Km ofapproximately 30 uM). When the number of IDO⁺ DCs is, large, a racemicmixture of 1MT is only partially effective in reversing IDO-mediatedinhibition of T cell proliferation. While this could in theory imply asecond, unrelated mechanism of suppression that happened to co-segregatewith IDO in our system, it more likely reflects the less-than-perfectefficiency of a racemic mixture of 1MT as an inhibitor of IDO. Theeffectiveness of 1MT was significantly enhanced if the DCs were matured(e.g., FIG. 2G), which may reflect a reduced potency of the IDO systemin mature DCs (as suggested by FIG. 2C), and the greater inherentstimulatory capacity of mature DCs.

Expression of IDO Protein without Constitutive Activity

FIGS. 2B-2E suggest that IDO may exist in both enzymatically-active andinactive forms. This type of regulation, in which the functionalenzymatic activity of a pre-formed pool of protein is turned on and offby post-translational events, is familiar in other enzyme systems.Examples include regulation of protein kinases by phosphorylation inkinase cascades, regulation of signal-transduction proteins byfarnesylation, and others. The shift between active and inactive statesis required for the proper biologic function of these enzymes. It is notyet known whether there exist post-translational modifications thatregulate IDO activity. However, it is known that factors which affectthe heme prosthetic group of IDO can markedly alter enzymatic activity(Thomas et al., J. Biol. Chem., 1994; 269:14457; Thomas et al., J.Immunol., 2001; 166: 6332) without necessarily affecting protein levels(Thomas et al., J. Immunol., 2001; 166:6332), so the possibility thatIDO could exist in both active and inactive forms is not withoutprecedent. Consistent with this, expression of immunoreactive IDOprotein without functional enzymatic activity has recently beendescribed in subsets of murine DCs (Fallarino et al., Int. Immunol.,2002; 14:65). FIG. 3D shows data consistent with the possibility thatactivation of Mφs with IFNγ may induce post-translational modificationsof IDO. This is suggested by the emergence of two additional proteinspecies on 2D-gel electrophoresis after activation with IFNγ. Thesespecies react with the anti-IDO antibody on western blot (FIG. 3D) andmigrate similarly to the major species in the SDS-PAGE dimension, butdisplay shifted isoelectric points. Such shifts in pI may be caused byalternate splicing, or, more commonly, may reflect post-translationalmodifications such as phosphorylation or acylation.

Thus, it appears that a constitutive presence in immature DCs ofimmunoreactive IDO protein, but with functional enzymatic activity beinginduced only after IFNγ activation, reflects an additional layer ofspecific, biologically relevant regulation of IDO. In this regard, itshould also be noted that, while flow cytometry failed to detectsignificant IDO in resting MCSF-derived Mφs (FIG. 2A), the moresensitive western blot analysis of detergent-solubilized whole-celllysates reveals a pool of constitutive IDO protein even in resting Mφs(FIGS. 3B and 3D), just as in immature DCs.

Example 2 A Small Population of Dendritic Cells in Tumor-Draining LymphNodes Mediates Dominant Immunosuppression Via Indoleamine2,3-Dioxygenase

The specific role of tolerogenic dendritic cells (DCs) in tumorimmunology remains unclear. In part, this is because the specificmolecular mechanisms by which DCs create tolerance are still poorlyunderstood (Moser, Immunity, 2003; 19:5). The present example focuses onthe immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) (Taylor etal., FASEB J., 1991; 5:2516). IDO is a tryptophan-degrading enzyme; itsexpression by cultured macrophages and DC allows them to inhibit T cellproliferation in vitro (Munn et al., J. Exp. Med., 1999; 189:1363; Hwuet al., J. Immunol., 2000; 164:3596; and Munn et al., Science, 2002;297:1867). Transfection of recombinant IDO into tumor cell lines confersthe ability to inhibit antigen-specific T cell responses in vitro(Mellor et al., J. Immunol., 2002; 168:3771), and protects immunogenictumor from rejection in vivo (Uyttenhove et al., Nat. Med., 2003;9:1269). Endogenous IDO has been implicated in maternal tolerance towardthe allogeneic fetus (Munn et al., Science, 1998; 281:1191), toleranceto self antigens in NOD mice (Grohmann et al., J. Exp. Med., 2003;198:153), and as a downstream effector mechanism for thetolerance-Inducing agent CTLA4-Ig (Grohmann et al., Nat. Immunol., 2002;3:985). Thus, IDO represents a potent endogenous immunoregulatory systemthat may be exploited by some tumors as a mechanism of immune evasion.

IDO can be expressed by a variety of human tumors and tumor cell lines(Uyttenhove et al., Nat. Med., 2003; 9:1269; Logan et al., Immunol.,2002; 105:478; and Friberg et al., Int. J. Cancer, 2002; 101:151). Thisobservation, combined with evidence from IDO-transfected tumors(Uyttenhove et al., Nat. Med., 2003; 9:1269), suggests that expressionof IDO by malignant cells may contribute to local immunosuppressionwithin tumors. As shown in Example 1, IDO is expressed by a populationof host cells found in certain tumor-draining lymph nodes (TDLNs),indicating that these cells represent a population of immunosuppressivehost DCs that are recruited by the tumor. In the present example, amurine tumor model was developed to isolate and characterize theIDO-expressing DCs from TDLNs was developed and used to studyIDO-dependent T cell suppression.

The present example demonstrates that tumor-draining lymph nodes in micecontain a population of plasmacytoid dendritic cells (pDCs) thatconstitutively express immunosuppressive levels of the enzymeindoleamine 2,3-dioxygenase (IDO). These cells expressed multiplemarkers of mature pDCs, but also co-expressed CD19 and pax5, suggestingderivation from a lymphoid B-cell progenitor. CD19⁺ pDCs comprised lessthan 0.5% of cells in tumor-draining lymph nodes (TDLN) but couldpotently and dominantly suppress CD8⁺ T cell responses in vitro, in anIDO-dependent fashion. Adoptive transfer of DCs from tumor-draininglymph nodes created T cell unresponsiveness and dominantimmunosuppression to a strong nominal antigen in naive,non-tumor-bearing hosts. These effects were abrogated by targeteddisruption of the IDO gene in the transferred DCs, or by administrationof the IDO inhibitor drug 1-methyl-tryptophan following adoptivetransfer. These results indicate that IDO-expressing DCs create a localmicroenvironment in tumor-draining lymph nodes that suppresses hostanti-tumor cytotoxic T cell responses.

Materials and Methods

Clinical studies. A series of samples from patients with malignantmelanoma were randomly selected, based on the following inclusioncriteria: radiographically mapped sentinel LN at the time of diagnosis;no metastases at presentation; and no further therapy given followinginitial surgical resection. Sentinel LN biopsies were stained for IDO byimmunohistochemistry, expression was graded by three pathologists asdescribed (Lee et al., Lab. Invest., 2003; 83:1457), and a consensusscore obtained. Patients were stratified into normal (grade 0) versusabnormal (grade 1+ or higher) expression, and compared by Kaplan-Meiersurvival analysis. Sentinel LN biopsies from patients with breast cancerwere selected from the archives of the Medical College of Georgia.Studies with human subjects were approved by the appropriateInstitutional Review Board.

Mouse tumor models. C57BL/6 mice (Jackson, Bar Harbor, Me.) wereimplanted with tumors in the anteriomedial thigh, using either 4×10⁴B16F10 (ATCC, Bethesda, Md.) or 1×10⁶ B78H1•GM-CSF. B78H1•GM-CSF cellsrecruit large numbers of APCs (Dranoff et al., Proc. Natl. Acad. Sci.USA, 1993; 90:3539) and were originally designed as a vaccine strategy.GMCSF-transfected tumors are immunogenic if lethally irradiated prior toinjection (Borrello et al., Hum. Gene Ther., 1999; 10:1983), but livetumors grow progressively and create systemic tolerance to tumorantigens (Bronte et al., J. Immunol., 1999; 162:5728). Thus,B78H1•GM-CSF represented an excellent model for our studies, since itrecruited many DCs but nevertheless provoke a protective immuneresponse. Tumors were 10-12 days after implantation, well prior to thestage of large, metastatic tumors (day 21) in which the hosts began toshow alteration in spleen and non-tumor-draining LNs. FACS analysisshowed that the pDCs recruited by B78H1•GM-CSF tumors werephenotypically identical to those recruited by B16F10 tumors, but withthe advantage that they could be recovered in quantities sufficient forfunctional analysis. Immunohistochemical studies of established B16F10and B78H1•GM-CSF tumors showed that neither expressed IDO in the tumorcells themselves. Studies, presented herein, using IDO-deficient hostsformally showed that the relevant IDO expression was in host-derivedAPCs, not the tumor cell lines themselves.

Immunohistochemistry. Immunohistochemistry was performed on humanmaterials as described in Example 1, following proteinase K antigenretrieval of formalin-fixed, paraffin-embedded sections. Mouse tissueswere fixed overnight in 10% formalin and paraffin embedded. Since theIDO epitope was not stable to prolonged storage in thin sections,staining was performed within 24 hours of sectioning. Staining wasperformed using a rabbit anti-mouse IDO polyclonal antibody (Mellor etal., J. Immunol., 2003; 171:1652). Cytocentrifuge preparations were madefrom cell suspensions following fluorescence-activated cell sorting,fixed for 10 minutes in 10% formalin, and stained within 24 hours.Controls for human and mouse staining included the anti-IDO antibodyneutralized with a molar excess of the immunizing peptide.

Flow cytometry and cell sorting. Single-cell suspensions of LNs wereobtained by teasing and disaggregation through a 40 micron mesh. Spleencells were obtained by ground-glass homogenization and hypotonic lysisof erythrocytes. Cells were stained by 4-color immunofluorescence, usingCD11c versus B220 versus CD19 versus a panel of other markers. Fcbinding was blocked using a commercial anti-CD16/CD32 cocktail (BDPharmingen, San Diego, Calif.). All acquisitions and sorts wereperformed using pulse-processing doublet discrimination. Antibodiesagainst the following antigens were from BD-Pharmingen: CD11c (cloneHL3), B220 (clone RA3-6B2), CD19 (clone 1D3), CD4 (clone H129.19), CD8α(clone 53.6.7), Ly6c (clone AL-21), CD45RA (clone H1100), MHC class II(anti-1-A^(b), clone 25-9-17), CD80 (clone 16-10A1), CD86 (clone GL1),H-2K^(b) (clone AF6-88.5), CD123 (clone 5B11), and CDI35/FLT3 (cloneA2F10.1). Anti-mouse CCR6 (clone 140706) was from R&D Systems(Minneapolis, Minn.). Anti-clonotypic antibody Ti98 against the BM3 TCRwas biotinylated and used as described (Tarazona et al., Int. Immunol.,1996; 8:351). All antibodies were used with isotype-matched negativecontrols. To adjust for differences in nonspecific binding between cellpopulations, each isotype control was gated on the specific populationof interest. Analytical flow cytometry was also performed on a 4-colorFACS-Caliber (Becton-Dickinson) with similar results.

T cell activation in MLR. BM3 responder T cells were prepared fromspleen by nylon-wool enrichment. Stimulators (sorted DCs orunfractionated TDLN) were mixed with 1×10⁵ BM3 responder cells in 200 ulIMDM at the ratios shown in each figure. After 3 days, proliferation wasmeasured by 4-hour thymidine incorporation assay. Where indicated,replicate groups of wells received 200 uM 1-methyl-[D]-tryptophan(Sigma-Aldrich, St. Louis, Mo.) or 250 uM [L]-tryptophan (Sigma). Toprepare a 100×1MT stock, a 20 mM solution was dissolved in 0.1 N NaOHand then adjusted to pH 7.4. All MLRs were performed in V-bottom culturewells (Nalge-Nunc, Rochester, N.Y.), as previously described in Example1, because the close cell-cell contact gave the maximum sensitivity toIDO-mediated suppression.

In these studies, it was important that the stimulator DCs not beirradiated, because initial validation studies showed that irradiationsignificantly altered the viability and functional attributes of IDO⁺pDCs. MLRs were thus “two-way” reactions. However, the small number ofsorted DCs used as stimulators contributed negligible proliferationcompared to the large population of TCR-transgenic responder cells.Since the relevant readout was dominant suppression, the two-way MLRdesign presented no problem in interpretation.

Quantitative real-time PCR. Total RNA was extracted from sorted cellsusing Trizol (Gibco-BRL, Gaithersburg, Md.). RNA was reverse-transcribedand amplified using the LightCycler real-time PCR system (Roche,Pleasanton, Calif.) with the RNA SYBR Green kit (Roche). All groups werecompared in the same run, and quantitated against a standard curve ofspleen RNA. Primers for mouse γ-actin were as follows. Sense wasGATGACGCAGATAATGTTT (SEQ ID NO:2) and antisense was TCTCCTTTATGTCACGAAC(SEQ ID NO:3), yielding a 290 basepair product. Primers for mouse CD19were as follows. Sense was GGCACCTATTATTGTCTCCG (SEQ ID NO:4) andantisense was GGGTCAGTCATTCGCTTC (SEQ ID NO:5), yielding a 218 basepairproduct). The primers for mouse pax5 were as follows. Sense wasGCATAGTGTCTACAGGCTCCG (SEQ ID NO:6) and antisense was GATGGGTTCCGTGGTGGT(SEQ ID NO:7), yielding a 299 basepair product. Conditions wereoptimized for each set of primers to give a linear standard curve over a1000-fold range, with a correlation coefficient of r greater than 0.99.An aliquot of each experimental sample was pre-screened to determineγ-actin message, then each sample was loaded to amplify an equivalentamount of γ-actin. For gels, RT-PCR was performed for the pre-determinedoptimum number of cycles yielding amplification in the linear range, andproducts resolved by formaldehyde gel electrophoresis. All primers gavesingle bands of the expected molecular weight.

Adoptive transfer studies. Recipient CBA mice (Jackson) were prepared byintravenous injection of 4×10⁷ BM3 splenocytes (termed CBA+BM3 mice). Ithas been previously shown that adoptively transferred BM3 T cells arestable in CBA hosts for over 100 days (Tarazona et al., Int. Immunol.,1996; 8:351). CD11c+ DCs were isolated from TDLNs, or from normal LNs ofC57BL/6 and CBA mice, using a Cytomation MoFlo high-speed cell sorter.Two aliquots of 1×10⁵ DCs were injected into each recipient,subcutaneously in each anteriomedial thigh (analogous to the position ofthe original tumor). After 10 to 12 days, recipient mice were euthanizedand the inguinal LNs (draining the sites of injection) and spleen (arepresentative distant site) removed for analysis. In some experiments,mice received 1-methyl-[D]-tryptophan (compound designation NSC 721782,Drug Development Group, Division of Cancer Treatment and Diagnosis,National Cancer Institute, Rockville, Md.) by continuous subcutaneousinfusion (5 mg/day) using implantable copolymer pellets as previouslydescribed (Munn et al., Science, 1998; 281:1191). Control mice receivedvehicle pellets alone.

IDO-Expressing Cells in Human TDLNs Predict a Poor Clinical Outcome

Screening studies of radiographically mapped tumor-draining (sentinel)lymph nodes from patients with breast carcinoma and malignant melanomademonstrated that a subset of patients had abnormal accumulation ofcells expressing IDO in sentinel nodes. Similar cells were also seen inregional LNs dissections from other solid tumors (colon, lung andpancreas, not shown). Melanoma was chosen for further analysis becauseof the availability of archived sentinel LNs with paired long-termclinical follow-up. In a retrospective study of 40 patients withmalignant melanoma, the presence of an abnormal number of IDO⁺ cells inthe sentinel LN at the time of diagnosis was found to be a significantlyadverse prognostic factor. None of these patients had detectablemetastases at the time of the biopsy, so recruitment of IDO⁺ cellsoccurred early in the course of the disease. In this example, a murinesystem to model these cells was developed and used to determine that theIDO-expressing cells in TDLNs represented a population ofimmunoregulatory APCs.

IDO-Expressing Cells in Murine TDLNs

The presence of IDO⁺ cells in TDLNs of mice with Lewis Lung Carcinomahas been previously reported (Friberg et al., Int. J. Cancer, 2002;101:151). With the present example, IDO⁺ cells were also found in TDLNsin the well-characterized B16F10 melanoma model. LNs were harvested 7-12days after tumor implantation, well before any detectable metastases tothe LNs. The accumulation of IDO⁺ cells occurred only in LNs drainingthe tumors; few or none were present in contralateral(non-tumor-draining) LNs from the same animals. Morphologically, theIDO⁺ cells were plasmacytoid mononuclear cells similar to those found inhuman nodes. However, the number of such cells recruited by B16F10tumors was lower than observed in heavily-infiltrated human LNs. Toincrease the number of APCs in the draining LNs, a subline of B16melanoma (clone B78H1) transfected with the cytokine GM-CSF (Huang etal., Science, 1994; 264:961) was also stained for IDO expression. GM-CSFmarkedly increases the number of APCs recruited into TDLNs (Dranoff etal., Proc. Natl. Acad. Sci. USA, 1993; 90:3539), and many primary humanmelanomas and other tumors constitutively express mRNA for GM-CSF(Mattei et al., Int. J. Cancer, 1994; 56:853; Smith et al., Clin. Exp.Metastasis, 1998; 16:655; Colasante et al., Hum. Pathol., 1995; 26:866;and Bronte et al., J. Immunol., 1999; 162:5728). B78H1•GM-CSF tumorsrecruited large number of IDO-expressing cells, comparable to heavilyinfiltrated humans LNs. As in B16F10, IDO expression was localized tothe draining node, and was not observed in contralateral nodes from thesame mice, nor in systemic sites such as spleen. Subsequent experimentstherefore used TDLNs from mice with B78H1•GM-CSF tumors

TDLNs Contain a Population of Suppressive Plasmacytoid DCs

Single-cell suspensions of TDLNs, or the paired contralateral LNs, wereused as stimulator cells in mixed leukocyte reactions (MLRs). Theresponder cells in MLRs were TCR-transgenic CD8⁺ T cells from BM3 mice,which recognize a nominal antigen (H-2K^(b)) constitutively expressed byall APCs from the C57BL/6 tumor-bearing hosts (Tarazona et al., Int.Immunol., 1996; 8:351). Preliminary validation studies confirmed thatexpression of the target antigen was high and comparable in DCs fromTDLNs and normal LNs. FIG. 5A shows that cells from TDLNs were poorstimulators of BM3 T cells, whereas cells from the contralateral(non-tumor-draining) LNs of the same animals were excellent stimulators(comparable to LN cells from non-tumor-bearing mice, not shown). Mixingexperiments (FIG. 5A, right panel) revealed that the failure ofresponder cells to proliferate in response to TDLN cells was due to adominant suppressor activity present in the TDLN cells.

Sorting experiments (FIG. 5B) revealed that a suppressor activity waspresent in a CD25⁺CD4⁺ fraction (2-3% of total cells) corresponding tothe Treg population known to be present in mice with B16 tumors(Sutmuller et al., J. Exp. Med., 2001; 194:823). Further sortingrevealed that a second, equally potent suppressor activity was alsopresent in the CD11c⁺B220⁺ fraction (1-2% of total cells) comprisingplasmacytoid DCs (pDCs). When these two suppressor populations wereremoved, the remaining 95-97% of TDLN cells stimulated excellentproliferation (as expected, since this fraction included all DCs otherthan pDCs, plus all B cells). Mixing experiments showed that suppressionby pDCs was dominant, and was also quite potent, since the 1-2% of pDCswas able to suppress responses stimulated by all of the other APCs.

Immunosuppression by pDCs is Mediated by IDO

To test whether T cell suppression by pDCs was mediated by IDO, MLRswere performed in the presence or absence of the IDO inhibitor1-methyl-tryptophan (1MT). FIG. 5C shows that 1MT blocked thesuppressive activity of pDCs, converting them into effective stimulatorsof T cell proliferation, and reversing their dominant suppression inmixing experiments. This was not due to any nonspecific activatingeffects of 1 MT on T cells, because the same T cells stimulated by thenon-suppressive “all other” fraction showed no enhancement by 1MT.

To further confirm that suppression by pDCs was mediated via IDO, tumorswere implanted in mice with a targeted disruption of the IDO gene(IDO-knockout mice). The pDCs isolated from TDLNs in these mice showedno suppressor activity (FIG. 5C, right panel). Further confirming thespecificity of 1MT for IDO, 1MT had no effect on MLRs stimulated byIDO-deficient pDCs.

Finally, the suppressive effect of pDCs was abrogated by addingsupraphysiologic, ten fold increased (10×), levels of L-tryptophan tothe MLR (FIG. 5D). This prevents IDO from depleting tryptophan, viasimple substrate excess, rather than by enzyme inhibition as with 1 MT,and circumvents IDO-mediated suppression of T cells (Munn et al., J.Exp. Med., 1999; 189:1363). Excess L-tryptophan abrogated suppressoractivity in a fashion similar to 1MT. Also as with 1 MT, there was noeffect of 10×L-tryptophan when pDCs were derived from IDO-KO hosts (FIG.5D, right panel).

Adoptive Transfer of DCs from TDLNs Creates Immunologic UnresponsivenessIn Vivo

Next, whether the DCs from TDLNs were sufficient to create immunologicunresponsiveness in vivo, independent of the original tumor, wasaddressed. CD11c⁺ DCs were isolated from TDLNs and adoptivelytransferred to new hosts. For these experiments, all of the DCs fromTDLNs were used, not just the pDCs, in order to accurately reflect themixed population found in TDLNs, and to ask whether stimulation ofsuppression would predominate in vivo. Ten days after adoptive transfer,T cells in the recipient host were tested for responsiveness to anominal antigen (H-2K^(b)) presented by the transferred DCs. Recipientswere allogeneic (H-2K^(b)-negative) CBA mice that had been pre-loadedwith a large cohort of H-2K^(b)-specific BM3 T cells.

FIG. 6A shows that adoptively transferred DCs induced selectiveaccumulation of antigen-specific BM3 T cells in inguinal LNs ofrecipient mice. This accumulation was comparable whether DCs werederived from TDLNs or from normal C57BL/6 LNs (both of which expressedH-2K^(b)). In contrast, DCs from antigen-negative CBA mice caused nosuch accumulation. In all groups, the number of BM3 T cells in thespleens (a site where BM3 T cells passively accumulate after injection)was similar, indicating that the initial loading was comparable.

Inguinal LN cells from recipient mice were assayed for functionalresponsiveness using MLRs stimulated by irradiated C57BL/6(H-2K^(b)-positive) splenocytes. FIG. 6B shows that T cells from micereceiving TDLN DCs displayed profound hyporesponsiveness to recallantigen stimulation, despite the presence of ample BM3 T cells (see,FIG. 6A). In contrast, T cells from control animals receiving normalC57BL/6 DCs (containing a comparable number of BM3 T cells) displayed abrisk MLR response. Mice receiving antigen-negative DCs also respondedwell in MLRs, confirming that the large cohort of pre-positionedtransgenic BM3 cells allowed a vigorous ex vivo response without theneed for any previous priming.

To test whether the state of acquired T cell unresponsiveness wascreated by IDO expression in the transferred TDLN DCs, recipient micewere treated with the IDO inhibitor 1 MT at the time of adoptivetransfer (control mice received pellets without 1 MT). After 10 days, Tcells were harvested and tested for responsiveness to antigen. RecallMLRs did not contain 1MT. FIG. 6C shows that administration of 1MTprevented the induction of T cell unresponsiveness in the recipients.The effect was specifically to block the acquisition ofunresponsiveness, not simply to enhance T cell responses in general,since 1MT had no enhancing effect on mice receiving normal DCs(right-hand panels). In all experiments, flow cytometry confirmed thatLNs from all groups contained comparable numbers of BM3 T cells, similarto FIG. 6A.

In the same experiments, the systemic response of T cells isolated fromspleens were also rested (FIG. 6C, lower panels). T cells from thisremote site showed a modest reduction in response compared to controls,which was prevented by 1 MT treatment, but T cells in the spleenretained significantly more responsiveness that T cells from drainingLNs. Thus, unresponsiveness was most profound in the LNs directlydraining the site of injection.

T Cells in Spleen Become Aware of Antigen Introduced on IDO⁺ DCs

It was not clear whether T cells in the spleen became “aware” ofantigens introduced on the transferred DCs. To address this question,the down regulation of clonotype-specific TCR on transgenic T cells wasexamined as a surrogate marker for antigen encounter (Tafuri et al.,Science, 1995; 270:630). Although descriptive, this helps make theimportant distinction between true immunologic “ignorance” (no encounterwith antigen) from encounter without activation. In vitro, it was foundthat BM3 T cells encountering antigen on TDLN cells showed a rapid andsustained down regulation of clonotype-specific TCR (FIG. 7A); incontrast, control BM3, stimulated by cells from normal LNs, retained TCRexpression. Thus, sustained down regulation of TCR served as a markerfor encounter with TDLN cells.

Applying this marker to adoptive transfer studies (FIG. 7A), it wasfound that BM3 T cells from hosts receiving TDLN DCs showed a uniformpattern of clonotype-specific TCR down regulation. In contrast, hostsreceiving normal DCs showed no down regulation. This was observed in theLNs draining the site of adoptive transfer, but was equally present inspleens from the same animals. Thus, even at distant sites such asspleen, responding T cells were not truly “ignorant” of antigen, but hadbeen affected the adoptively-transferred DCs (even though the affectedcells were not anergic); and this effect was specific for TDLN DCscompared to normal DCs. When TDLN DCs were derived from IDO-knockoutmice, there was little TCR down regulation following adoptive transfer(FIG. 7B), suggesting that most of the down regulation wasmechanistically due to IDO expression by the TDLN DCs. A similar effectwas seen when 1MT was administered following adoptive transfer, alsosupporting the role of IDO. IDO-deficient TDLN DCs also did not createfunctional T cell unresponsiveness in the recipients (FIG. 7B, rightpanel), similar to recipient mice treated with 1MT (FIG. 6C).

IDO⁺ DCs Create Secondary Suppressor Mechanisms that are Independent ofIDO

Next, it was addressed whether the T cell unresponsiveness created byadoptive transfer was due to intrinsic anergy of the responding T cells,or to some form of active suppression. Mixing experiments were performedusing two groups of responder cells. One group of responder cells wascontrol T cells isolated from CBA+BM3 recipients following adoptivetransfer of normal DCs (fully responsive); and the second group ofresponder cells was T cells from the same mice following adoptivetransfer of TDLN DCs (unresponsive). Mixing experiments (FIG. 7C) showedthat the “unresponsive” cells entirely suppressed proliferation by theotherwise competent population (FIG. 7C, arrow). This was thus notconsistent with simple anergy on the part of the unresponsive cells, butinstead indicated a component of active, dominant suppression.

To test whether this suppression was mediated by IDO, recall MLRs wereperformed in the presence or absence of 1MT. FIG. 7C (right panel) showsthat 1MT had no effect on suppression in recall MLRs, implying that IDOwas not the mechanism of suppression. Thus, while the creation ofunresponsiveness was absolutely dependent on IDO during its inductionphase (FIGS. 6C and 7B), the unresponsive state was maintained, at leastin part, by additional, IDO-independent mechanisms.

IDO-Mediated Suppression Segregates with a Small Subset of CD19⁺ DCs

The preceding studies showed that some IDO-expressing cell type in TDLNswas responsible for creating immunologic unresponsiveness in vivo. Tobetter define the specific DC subset, more detailed sorting experimentswere performed. The experiments in FIG. 5 had shown that IDO-mediatedsuppressor activity segregated with the plasmacytoid (B220⁺) DCfraction. Further phenotyping studies of this population showed it to beheterogeneous with respect to a number of markers, in particularexpression of CD19. DCs from TDLN cells were sorted into CD19⁺ andCD19^(NEG) fractions of pDCs, as well as conventional B220^(NEG) DCs,following the schema shown in FIG. 8. Immunohistochemistry on the sortedcells revealed that all three populations contained at least some cellswith immunoreactive IDO. To quantitatively measure the IDO-mediatedsuppressor activity associated with each subset, the fractions wereanalyzed as stimulators in MLRs with and without 1 MT. Control MLRsreceived B cells from the same sorting run, which were good stimulatorswith no suppressor activity. FIG. 8 shows that virtually all of theIDO-mediated suppressor activity segregated with the novel CD19⁺ DCfraction. When these CD19⁺ DCs were removed, the remaining DCs showedminimal suppression (FIGS. 8B and 8C).

Despite comprising almost all of the IDO-mediated suppression, CDI9⁺pDCs were a small fraction of total LN cells. In a total of 13experiments (analyzing 2-6 pooled TDLNs each), the total CD11c⁺ DCs werefound to comprise 1-1.5% of cells. Of these, CD19⁺ pDCs comprised 31±15%of DCs. Thus, the potent IDO-induced suppression seen in FIG. 5B,capable of dominantly inhibiting the proliferation stimulated by all ofthe other nonsuppressive DCs and B cells in mixing experiments, wasmediated by 0.3-0.5% of total LN cells.

CD19⁺ DCs Show a Phenotype of Mature Plasmacytoid DCs

FIG. 9A shows immunophenotyping of cells from TDLNs, gated first on theCD11c⁺ DCs, then further gated into CD19⁺ and CD19^(NEG) fraction. Othermarkers (including B220) were analyzed in the third and fourth colors.FIG. 9A shows that all of the CD19⁺ DCs in TDLNs expressed B220,consistent with their being a subset of pDCs (although not all of theB220⁺ pDCs expressed CD19). Many of the CD19⁺ DCs also expressed CD4and/or CD8a, both of which can be found on pDCs (and both of whichserved to unambiguously distinguish CD19⁺pDCs from B cells). Many of theCD19⁺ DCs also expressed Ly6c and CD45RA, which are markers associatedwith murine plasmacytoid DCs (O'Keeffe et al., J. Exp. Med., 2002;196:1307; and Martin et al., Blood, 2002; 100:383). CD19⁺ DCs alsouniformly expressed the receptor tyrosine kinase Flt3 (CD135), which isexpressed on DCs but not on mature B cells (Karsunky et al., J. Exp.Med., 2003; 198:305).

To confirm that the apparent expression of CD19 was authentic, mRNA forCD19 was measured by quantitative RT-PCR. The upstream transcriptionfactor pax5 was also measured, since expression of CD19 is obligatelydependent on expression of pax5 (Nutt et al., EMBO J, 1998; 17:2319; andMikkola et al., Science, 2002; 297:110). FIG. 9B confirms that cells ofthe CD19⁺ pDC fraction expressed mRNA for both CD19 and pax5.Quantitatively, these genes were present at somewhat lower levels thanin mature B cells (normalized to γ-actin), but they were expressed atmuch higher levels than in either the CD19^(NEG) fraction of pDCs, orthe B220^(NEG) conventional DCs, and were thus unambiguously positive.

In other experiments CD19⁺ pDCs of the same phenotype (CD11c⁺ CD19⁺,B220⁺, Ly6c⁺, CD45RA⁺) were also found in normal LNs fromnon-tumor-bearing hosts, as well as in the contralateral LNs of micewith B78H1•GM-CSF tumors, and in TDLNs from mice with B16F10 tumors. Inthe normal LNs they comprised 18±6% of total DCs (n=6 experiments),compared to 31±15% of total DCs in B78H1•GM-CSF TDLNs.

CD19⁺ pDCs TDLNs uniformly expressed high levels of MHC class IImolecules, and costimulatory molecules CD80 and CD86, suggestive of amature phenotype (FIG. 9C). These were expressed at levels equal to orgreater than the CD19^(NEG) DCs from the same LN, many of which wereimmature, as shown. In different TDLNs, the CD19^(NEG) subset of DCsshowed significant variability in the number of mature versus immatureDCs; however, all of the CD19+ pDCs were invariably mature.

In addition to maturation markers, the CD19⁺ pDCs also expressed twomarkers observed in the human system. As shown in Example 1, CD123(IL3Ra) and the chemokine receptor CCR6 segregate closely with IDOexpression in human monocyte-derived DCs and macrophages. In thisexample, it was found that in murine TDLNs, expression of CD123 and CCR6preferentially associated with the CD19+ subset of DCs in TDLNs (FIG.9D). In contrast, the majority of CD19^(NEG) DCs were low or negativefor these markers (FIG. 9D), as were the majority of B cells, which wereless than 20% positive for CCR6 and less than 2% positive for CD123.

Discussion

The current example identifies a small population of immunoregulatoryDCs in TDLNs that are capable of mediating dominant immunosuppression invitro, and creating profound immunologic unresponsiveness in vivo. Ofthe limited number of studies assessing DCs from TDLNs, most havereported a defect in their ability to stimulate T cells (Vicari et al.,J. Exp. Med., 2002; 196:541; Almand et al., Clin. Cancer Res., 2000;6:1755; and Yang et al., J. Clin. Invest., 2003; 111:727). However, ithas been unclear whether this was due to immaturity of the DCs, or tosome form of active suppression. This example shows that one subset ofDCs in TDLNs can create potent active suppression, mediated via themolecular mechanism of IDO.

The suppression created by IDO-expressing DCs was profound, but it wasfundamentally local. First, the expression of IDO itself was strictlylocalized, being found in the LNs draining tumors, but not in other LNsfrom the same animal. Second, adoptive transfer of DCs from these nodescreated complete T cell unresponsiveness in the new nodes draining thesite of injection, while T cells in spleens of the same animals remainedreactive. An analogous dichotomy has been observed in tumor-bearinghosts, where tumors may be locally tolerated despite the systemicpresence of competent, tumor-specific T cells (Wick et al., J. Exp.Med., 1997; 186:229; Speiser et al., J. Exp. Med., 1997; 186:645; andNguyen et al., J. Exp. Med., 2002; 195:423). The fact that tumors inthis situation grow unchecked, ultimately killing their hosts,emphasizes the point that immunosuppression does not have to be systemicin order to be effective. Functionally, if all the draining LNs of allthe sites of tumor create local unresponsiveness, the tumor is de factotolerated.

In this regard, it is relevant to note that the immunosuppressioncreated by IDO-expressing DCs was dominant. This was shown by in vitromixing experiments, in which a small population of CD19⁺ pDCs from TDLNscould completely suppress T cell responses, despite the presence of manyother stimulatory APCs (for example, see FIG. 5). It remains to beelucidated how such a small population of IDO⁺ DCs can effectivelycontrol a large T cell response, but this same phenomenon has beenobserved in other studies of IDO-expressing DCs (Grohmann et al., J.Immunol., 2000; 165:1357; and Mellor et al., J. Immunol., 2003;171:1652).

This example demonstrated that the adoptive transfer of TDLN DCs in vivowas able to create local immunologic unresponsiveness to a strongnominal antigen (H-2K^(b)), even in the presence of a large cohort ofantigen-specific, TCR-transgenic T cells. This local effect wasaccompanied by a systemic effect on all transgenic T cells, as shown bysustained down regulation of TCR expression. It has been suggested thatsuch TCR down regulation may serve as a marker for anergy (Benson etal., J Clin. Invest., 2000; 106:1031; and Tafuri et al., Science, 1995;270:630), but anergy alone could not account for all of theimmunosuppression created by the TDLN DCs. Mixing experiments revealedthat there was also a potent component of active suppression, capable ofinhibiting other, fully competent responder T cells in vitro (see FIG.7C).

This secondary suppressor activity was not itself mediated by IDO, butit was obligately dependent on expression of functional IDO by TDLN DCsfor its creation. Thus, the IDO expressed by TDLN DCs elicited asecondary mechanism to amplify and sustain its immunosuppressive effect.

These observations are consistent with findings in other studies usingCD8a⁺ DCs from spleen, activated in vitro to express IDO. Adoptivetransfer of even small numbers of such IDO-expressing DCs was capable ofcreating systemic unresponsiveness to antigen (Grohmann et al., J.Immunol., 2000; 165:1357; and Fallarino et al., Nat. Immunol., 2003;4:1206-12).

In the present system, the immunosuppression mediated by IDO-expressingDCs was an active process, not merely a passive failure of immature DCsto stimulate T cells. It is well established that immature DCs can betolerogenic (Hawiger et al., J. Exp. Med., 2001; 194:769; and Probst etal., Immunity, 2003; 18:713), which has been attributed to their failureto provide adequate costimulation. However, it has been morecontroversial whether certain DCs may be tolerogenic even when mature(Moser, Immunity, 2003; 19:5). The CD19⁺ pDCs found in TDLNs appearedphenotypically mature (positive for CD80 and CD86, high MHC-IIexpression, and excellent stimulators of T cell proliferation when IDOwas blocked). Despite this, they were also actively suppressive invitro, as shown by mixing experiments. In order to clearly demonstratethe active nature of this suppression in vivo, the present adoptivetransfer studies were designed so that vigorous T cell activation wasthe default response. A strong alloantigen (H-2K^(b)) was employed,constitutively expressed on all transferred DCs, recognized by a massivepopulation of pre-positioned TCR-transgenic T cells (up to 40% ofrecipient CD8⁺ T cells). Because of this large “pre-expanded” clone of Tcells, there was no need for any initial priming step in order to see arobust proliferative response in MLR assays. Even in this system, DCsfrom TDLNs were able to create acquired unresponsiveness to the H-2K^(b)antigen, in an IDO-dependent fashion.

Fractionation studies of TDLN cells showed that virtually all of theIDO-mediated suppressor activity segregated with a novel population ofCD19⁺ DCs. CD19, and its obligate transcription factor pax5, are markersof the B cell lineage (Fearon and Carroll, Ann. Rev. Immunol., 2000;18:393; and Nutt et al., Nature, 1999; 401:556), so expression of thesegenes suggests derivation from a B-lineage precursor (or a commonlymphoid progenitor cell). Consistent with this possibility, it is knownthat early CD19⁺ pro-B cells can give rise to DCs in vitro (Bjorck andKincade, J. Immunol., 1998; 161:5795; and Izon et al., J. Immunol.,2001; 167:1387). Recent analyses of both human and murine plasmacytoidDCs suggests a B-cell origin for a subset of pDCs (Rissoan et al.,Blood, 2002; 100:3295; and Corcoran et al., J. Immunol., 2003;170:4926); in mice, up to one-third of DNA from murine splenicplasmacytoid DCs was found to show D-J rearrangement of the IgH locus(Corcoran et al., J. Immunol., 2003; 170:4926). Despite this link,previous studies have failed to identify the CD19⁺ subset of pDCs. Inpart, this may be because many studies have specifically excluded theCD19⁺ cells, either by depletion or by back-gating (O'Keeffe et al., J.Exp. Med., 2002; 196:1307; and Asselin-Paturel et al., Nat. Immunol.,2001; 2:1144). Even if recognized as DCs, it would not be obvious thatthe CD19⁺ cells were tolerogenic, because resting CD19⁺ DCs do notconstitutively express IDO, and are not suppressive. It was only in thecontext of TDLNs that the regulatory attributes of these cells becameevident.

The key difference between TDLNs and normal LNs was not the presence orabsence of CD19⁺ DCs, as they were present in both, although at highernumbers in the TDLNs, but rather the fact that CD19⁺ DCs from TDLNsconstitutively expressed IDO. It has been previously shown that theIDO-inducing agent CTLA4-Ig up regulates IDO preferentially in the B220⁺and CD8a⁺ DC subsets (Mellor et al., J. Immunol., 2003; 171:1652). Priorto CTLA4-Ig treatment, these DCs were not inhibitory, but they became soafter IDO was induced (Mellor et al., J. Immunol., 2003; 171:1652),indicating that some factor in TDLNs acts to induce constitutiveexpression of IDO in CD19+ pDCs. This factor might be amicroenvironmental signal, such as a cytokine, found selectively inTDLNs. Alternatively, it has recently been shown that CD25⁺CD4⁺ Tregscan induce IDO in DCs via expression of CTLA4 (Fallarino et al., Nat.Immunol., 2003; 4:1206-12). Thus, it is possible that TDLNs mightcontain a population of Tregs that triggers IDO. Elucidating theupstream factors responsible for the constitutive induction of IDO inTDLNs may offer significant insight into how tumors evolve to exploitthe IDO mechanism.

Example 3 1-Methyl-[D]-Tryptophan, a Novel, Small-Molecule,Orally-Bioavailable Immune Modulator for Use in Cancer Immunotherapy

Tumors actively create a state of tolerance toward their own antigens.This pathologic situation allows the tumors to escape from host immunesurveillance and also imposes a barrier to effective anti-tumorimmunotherapy. One molecular mechanism by which tumors may inhibitimmune responses is via the immunosuppressive enzyme indoleamine2,3-dioxygenase (IDO). IDO degrades the amino acid tryptophan, and thisacts to inhibit T cell responses. The small-molecule1-methyl-D-tryptophan (1MT) acts as an inhibitor of IDO enzyme activityin vitro, and is capable of preventing IDO-mediated immunosuppression invivo. 1 MT thus acts as an immune-enhancing agent in a variety of animalmodels where IDO limits or suppresses immunologic responses. In thisexample, administration of 1MT to tumor-bearing hosts in conjunctionwith low-dose chemotherapy or radiation shows synergisticimmune-mediated anti-tumor effect. 1MT thus represents a novelsmall-molecule, orally-bioavailable immune modulator for use in cancerimmunotherapy.

1 MT targets a previously unrecognized immunosuppressive pathway. Thisendogenous pathway may limit the efficacy of current immunotherapyapproaches. One application of 1MT is as a vaccine adjuvant, sinceanti-tumor vaccines have shown occasional encouraging responses buttheir overall response rates remain limited (Yu and Restifo, Journal ofClinical Investigation 2002; 110:289-294). Thus, adding 1MT as anadjuvant to an existing (Phase II) anti-tumor vaccine would be ethicaland appropriate. Further, it is likely unnecessary to supply exogenoustumor antigen in the form of a vaccine. Chemotherapy and radiationalready release large amounts of antigen from dying tumor cells, butthese do not normally generate a useful immune response. 1MT will allowimmune responses to such antigens.

Comparison of D- Versus L-Isomers of 1MT

Cultured human dendritic cells (DCs) enriched for IDO expression wereprepared as described by Munn et al., Science 2002; 297:1867-1870. DCswere used as stimulators in allogeneic mixed-leukocyte reactions (MLRs),with allogeneic lymphocytes as responder cells. The ability of 1MT toinhibit IDO-mediated suppression is measured as the amount of T cellproliferation. FIG. 10 shows that the D isomer was significantly moreeffective than the L isomer at reversing IDO-mediated suppression.

IDO-Expressing Cells in Human Tumor-Draining Lymph Nodes

Using a polyclonal antibody against human IDO, abnormal accumulation ofIDO⁺ cells was seen in many tumor-draining (sentinel) lymph nodes frompatients with malignant melanoma (Example 2 and as described in Lee etal., Laboratory Investigation 2003; 83:1457-1466). A retrospectiveanalysis of sentinel nodes from 40 patients with locally-confineddisease at diagnosis showed the presence of these IDO⁺ cells was anadverse prognostic factor, even prior to detectable metastasis.

IDO⁺ Cells are Selectively Recruited into Mouse Tumor-Draining LymphNodes

Tumor-draining lymph nodes from C57BL/6 mice implanted with B16F10melanoma cells (day 14) showed significant accumulation of IDO⁺ cellscompared contralateral lymph nodes from the same animals.Phenotypically, the IDO⁺ cells were mostly CD11c⁺B220⁺ “plasmacytoid”dendritic cells. To obtain larger lymph nodes for functional studies,B78H1.GMCSF, a GMCSF-transfected sub-line of B16 was also used (Huang etal., Science 1994; 264:961-965; and Borrello et al., Human Gene Therapy1999; 10:1983-1991). These tumors recruited the same IDO⁺ cells, butyielded quantitatively more cells for study. GMCSF-transfected celllines recruit large numbers of DCs (Dranoff, Immunological Reviews 2002;188:147-154), but as viable tumors they have been found to inducetolerance instead of spontaneous immunity (Bronte et al., J. Immunol.1999; 162:5728-5737).

IDO-Mediated Suppression Segregates with the Plasmacytoid DCs

FIG. 5 shows IDO-expressing suppressive APCs are present intumor-draining LNs. Pooled draining LN cells (4 nodes) were stained andfractionated by 4-color Mono cell sorting for the two populations shownin the schematic of FIG. 5B. All other cells were collected in a thirdfraction. Each fraction was then used as stimulators in MLRs, using50,000 BM3 T cells as responders. The number of stimulators used in eachMLR was adjusted to be the same as would have been present in 50,000cells of the original LN preparation, based on the measured percentageof each sorted fraction. Thus, 500 cells of the sorted B220⁺ CD11c⁺fraction were added per well, and 1500 cells of the CD25⁺ CD4⁺ fraction,while 48,000 cells of the “all other” fraction were used. Replicate MLRswere performed with or without 1MT, as shown. Tumor-draining LN cellsfrom a wild-type (IDO-sufficient) C57BL/6 host and tumor-draining LNcells from an IDO-knockout host, showing no IDO-mediated inhibition (butwith inhibition by Tregs intact) are shown in FIGS. 5C and 5D. Thearrows in FIG. 5C indicate the IDO-mediated (1MT-sensitive) component ofinhibition.

Cells were harvested from tumor-draining lymph nodes (day 12) and usedas APCs in allogeneic mixed-leukocyte reactions (MLRs). Responder Tcells were taken from “BM3” TCR-transgenic mice (CD8⁺, recognizingH-2K^(b) (Tarazona et al., International Immunology 1996; 8:351-358)).Cells were sorted into B220⁺ CD11c⁺ plasmacytoid DCs, and a separateCD25⁺CD4⁺ fraction of regulatory T cells. It was important to separateout these Tregs, otherwise they would nonspecifically inhibitproliferation in the readout MLRs. The third fraction collectedcomprised the remaining 96% of the lymph-node cells; these cellsstimulated good T cell proliferation, and there was no enhancement by1MT (thus confirming that 1MT had no nonspecific stimulatory effect inthe absence of IDO). The plasmacytoid DC fraction was potentlyinhibitory, and this was fully reversed by 1MT. Mice with a targeteddeletion of IDO were as described in Mellor et al., J. Immunol. 2003;171:1652-1655 9. These IDO-knockout mice showed no suppressor activityby plasmacytoid DCs, and no effect of 1MT, thus confirming that themolecular target of 1MT was indeed IDO.

Effect of 1MT on Established Tumors, and Synergy with Radiation orCyclophosphamide

B16F10 melanomas were implanted in C57BL/6 mice, then 7 days later micewere treated with 1MT or vehicle control (administered by SQ continuousinfusion using implantable co-polymer pellets (Munn et al., Science1998; 281:1191-1193)). The initial studies used the DL racemic form of1MT, at a total dose of 20 mg per mouse per day. In thisestablished-tumor model, 1MT alone had no effect. Not unexpected, giventhat the tumor had already been allowed to create tolerance in the host.However, when the established host/tumor milieu was transientlyperturbed by a single dose of total-body radiation (500 cGy) orcyclophosphamide (150 mg/kg×1 dose), 1 MT acted synergistically withboth interventions, to significantly reduce tumor growth (FIGS. 11A and11B). Although the combination was not curative, this degree of growthdelay was comparable to that seen with other immunologic interventionsin this aggressive tumor model (van Elsas et al., J Exp Med 1999;190(3):355-66; and Kotera et al., Cancer Research 2001;61(22):8105-8109). Identical experiments performed in immunodeficient(RAG1-knockout) hosts showed no enhancing effect of 1 MT overcyclophosphamide alone (FIG. 11C), indicating that the effect of 1MT wasentirely immunologically mediated. Finally, the [D]-isomer of 1 MT wasfound to be effective at one quarter the dose used for the racemicpreparation (FIG. 11D).

The radiation studies shown in FIG. 11A were replicated using a similarexperimental design. The pattern of response observed in these studieswas similar to that shown in FIG. 11A, with 1MT alone having no effect,but with the combination of the [D]-isomer of 1MT and radiation showingenhanced effect over radiation alone.

Pharmacokinetics

Comparative pharmacokinetic studies were initiated to characterize thein vivo disposition of the stereoisomers of 1-MT when administered bydifferent routes, to determine the oral bioavailability, and to definethe plasma concentrations associated with effective dosing regimens.Studies were conducted in conventional mice after IV, PO and SC doses of50 mg/kg, and IP doses of 25 mg/kg, 50 mg/kg, and 100 mg/kg. Inaddition, plasma concentrations of I-MT were determined in nude micewith implanted timed-release pellets designed to release the DL isomerfor 7 days, and the D isomer for 14 days.

Peak plasma concentrations of D or L 1-MT were attained 2 hours after POadministration. The maximum concentration observed for the L isomer wasapproximately seven times greater than that for the D isomer. The AUC0-∞for the L isomer was also substantially greater (9 fold) than that forthe D isomer. Terminal disposition phase half-lives were similar for thetwo isomers, and also similar to those found after IV administration.The oral bioavailability for D and L 1-MT were 64% and 105%,respectively.

The in vivo disposition of the D and L isomers of 1-MT are remarkablydifferent considering their nearly identical chemical structures.Analysis of their plasma pharmacokinetics suggests that the basis forthe difference is a rapid partitioning of the D isomer into tissuescompartments other than the plasma, while the L isomer is morepredominantly distributed in the plasma. As a consequence, the AUC0-∞observed for L 1-MT were from 5 to 9 times greater than those found forD 1-MT with equivalent routes of administration and doses. Given thecomparatively lower plasma levels of the D isomer, while tempting tospeculate regarding where the D isomer does reside, the experimentaldesign of these studies did not encompass examination of the tissue orcellular distribution of 1-MT. However, one possible explanation toaccount for the relatively greater apparent volume of distribution ofthe D isomer is a higher intracellular localization, raising thepossibility of a unique cellular transport mechanism for D 1-MT notshared by L1-MT. Cellular uptake studies will be performed with the twoisomers to address this pivotal issue.

Both isomers were absorbed efficiently following IP, SC and POadministration, and the plasma pharmacokinetics were comparableregardless of the route of administration, indicating that any of theroutes studied are equally suitable for efficacy testing. The oralbioavailability of the D isomer (64%) was somewhat lower than thatobserved when it was given by the intraperitoneal (IP) or subcutaneous(SC) route, and also lower than the oral bioavailability of L 1-MT.

Pilot Studies to Address the Tissue Biodistribution of the D-Isomer

Mice were given 1MT as the DL racemic mixture (10 mg/day, SQ implantedcopolymer pellets, 72 hour infusion), then tissues were harvested,extracted, and tissue levels of the respective D and L isomersdetermined by generation of diastereomeric isoindoyl derivatives withOPA and Boc-L-Cys (Hashimoto et al., J Chromatography 1992; 582:41-48).Initial results showed at least equal, and possibly preferential,accumulation of the D isomer in liver, with equivalent distribution of Dand L isomers into muscle and spleen. The results noted with spleen areparticularly relevant because it is a lymphoid organ, and thus likely tobe similar to tumor-draining lymph nodes. Initial results also showedlower levels of the D isomer in plasma and in brain, suggesting that theD isomer may not cross the blood-brain barrier efficiently. Thus, thelower plasma levels and shorter intravascular half-life do notnecessarily imply lower levels in the relevant tissues.

Discussion

1-MT represents the lead compound in a new class of immunomodulatoryagents, designed to block immunosuppression mediated by IDO. Manytumors, under the selection pressure of host immune surveillance, haveevolved some means to exploit the tolerogenic activity of IDO, eitherdirectly, through expression of IDO by tumor cells, or by recruitingIDO-expressing APCs to induce systemic tolerance. Thus, 1MT will beindicated as an immunologic adjuvant in combination with anti-tumorvaccines, including peptide, cell-lysate, or dendritic cell-basedvaccines, and as an immunomodulatory agent in combination withchemotherapy or radiation. 1MT is a lead compound, representative of anew class of immunomodulatory drugs designed to inhibit the IDO pathway.It is one of a number of small-molecule, orally-bioavailableimmunostimulatory agents.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. The foregoing detaileddescription and examples have been given for clarity of understandingonly. No unnecessary limitations are to be understood therefrom. Theinvention is not limited to the exact details shown and described, forvariations obvious to one skilled in the art will be included within theinvention defined by the claims.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

SEQUENCE LISTING FREE TEXT

SEQ ID NO:1 peptide; human indoleamine-2,3-dioxygenase (IDO)SEQ ID NO:2-7 Synthetic oligonucleotide primers

1-43. (canceled)
 44. A method of enhancing the efficacy of a vaccine ina subject, the method comprising administering to said subject asufficient quantity of said vaccine and D-1-methyl tryptophan (D1MT).45. The method of claim 44, wherein said vaccine comprises an antigenicprotein.
 46. The method of claim 44, wherein said vaccine is a tumorvaccine.
 47. The method of claim 46, wherein said tumor vaccinecomprises a tumor cell comprising a tumor antigen.
 48. The method ofclaim 47, wherein the tumor antigen is from a cancer selected from thegroup consisting of melanoma, colon cancer, pancreatic cancer, breastcancer, prostate cancer, lung cancer, leukemia, brain tumors, lymphoma,sarcoma, ovarian cancer, and Kaposi's sarcoma.
 49. The method of claim46, wherein said tumor vaccine comprises a genetically modified tumorcell, or a genetically modified tumor cell line.
 50. The method of claim44, wherein the vaccine is an anti-viral vaccine.
 51. The method ofclaim 50, wherein said vaccine comprises a viral antigen.
 52. The methodof claim 51, wherein said viral antigen is from Human ImmunodeficiencyVirus (HIV) or cytomegalovirus.
 53. The method of claim 44, wherein saidvaccine is an anti-bacterial vaccine.
 54. The method of claim 53,wherein said vaccine comprises a bacterial antigen.
 55. The method ofclaim 54, wherein said bacterial antigen is from Mycobacterium leprae,Mycobacterium tuberculosis, Listeria monocytogenes, or Toxplasma gondii.56. The method of claim 44, wherein said vaccine is administered priorto, concurrently with, or after the D-1-methyl tryptophan (D1MT). 57.The method of claim 44, wherein the D-1-methyl tryptophan (D1MT) isformulated for oral delivery.
 58. The method of claim 57, wherein theD-1-methyl tryptophan (D1MT) is formulated as a powder, capsule, tabletor liquid.
 59. The method of claim 44, wherein the 1-methyl-D-tryptophanis substantially free of the L isomer.
 60. A method to increase theimmune response elicited by a vaccine, the method comprisingadministering to a patient said vaccine plus D-1-methyl tryptophan(D1MT).
 61. The method of claim 60, wherein said vaccine comprises anantigen in combination with an adjuvant.